User Research

Here is a collection of research, written by users and other third-party members, which cite the usage or methods present in the PEAKS software.

User Published Research Debreuil J, Tambutté E, Zoccola D, Deleury E, Guigonis J-M, Samson M, Allemand D, and Tambutté S. Molecular cloning and characterization of the first organic matrix protein from the sclerites of the red coral, Corallium rubrum. J Proteome Res. 2012 Mar 30. [Epub ahead of print].
We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an EST library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.
User Published Research Pereira-Medrano A, Margesin R, Wright P. Proteome characterization of the unsequenced psychrophile Pedobacter cryoconitis using 15N metabolic labeling, tandem mass spectrometry, and a new bioinformatic workflow. Proteomics 2012, 12, 775–789. March 2012.
Organisms without a sequenced genome and lacking a complete protein database encounter an added level of complexity to protein identification and quantitation. De novo sequencing, new bioinformatics tools, and mass spectrometry (MS) techniques allow for advances in this area. Here, the proteomic characterization of an unsequenced psychrophilic bacterium, Pedobacter cryoconitis, is presented employing a novel workflow based on 15N metabolic labelling, 2DE, MS/MS, and bioinformatics tools. Two bioinformatics pipelines, based on nitrogen constraint (N-constraint), ortholog searching, and de novo peptide sequencing with N-constraint similarity database search, are compared based on proteome coverage and throughput. Results demonstrate the effect of different growth temperatures (1°C, 20°C) and different carbon sources (glucose, maltose) on the proteome. Seventy-six and 69 proteins were identified and validated from the glucose- and maltose-grown bacterium, respectively, from which 21 and 22 were differentially expressed at different growth temperatures. Differentially expressed proteins are involved in stress response and carbohydrate metabolism, with higher expression at 20°C than at 1°C, while antioxidants were upregulated at 1°C. This study provides an alternative workflow to identify, validate, and quantify proteins from unsequenced organisms distantly related to other species in the protein database. Furthermore, it provides further understanding on bacterial adaptation mechanisms to cold environments, and a comparative proteomic analyses with other psychrophilic microorganisms.
User Published Research Zhang X, Petruzziello F, Zani F, Fouillen L, Andren PE, Solinas G, Rainer G. High Identification Rates of Endogenous Neuropeptides from Mouse Brain.. J Proteome Res. 2012 Mar 30. [Epub ahead of print].
Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 μL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC-MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.
User Published Research Vermeij WP, Florea BI, Isenia S, Alia A, Brouwer J, and Backendorf C. PROTEOMIC IDENTIFICATION OF IN VIVO INTERACTORS REVEALS NOVEL FUNCTION OF SKIN CORNIFICATION PROTEINS. Journal of Proteome Research. 2012 Apr 23.
Protection against injurious external insults and loss of vital fluids is essential for life and is in all organisms, from bacteria to plants and humans, provided by some form of barrier. Members of the small-proline-rich (SPRR) protein family are major components of the cornified cell envelope (CE), a structure responsible for the barrier properties of our skin. These proteins are efficient reactive oxygen species (ROS) quenchers involved not only in the establishment of the skin’s barrier function but also in cell migration and wound healing. Here, a proteomic analysis of in-vivo SPRR-interacting proteins confirmed their function in CE-formation and ROS-quenching and also revealed a novel unexpected role in DNA-binding. Direct in-vitro and in-vivo evidence proved that the DNA-binding capacity of SPRRs is regulated by the oxidation state of the proteins. At low ROS levels, nuclear SPRR is able to bind DNA and prevent ROS-induced DNA damage. When ROS levels increase SPRR proteins multimerize and form an effective antioxidant barrier at the cell periphery, possibly to prevent the production or infiltration of ROS. At even higher ROS exposure DNA-binding is restituted. A molecular model explaining how the intracellular oxidation state of SPRRs likely influences their selective protective function is provided.
User Published Research Fields C, Mallee P, Muzard J, Lee G. Isolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separation. Food Chemistry. 2012 Mar 30. [Epub ahead of print].
Soybean proteins offer exceptional promise in the area of cancer prevention and treatment. Specifically, Bowman-Birk inhibitor (BBI) has the ability to suppress carcinogenesis in vivo, which has been attributed to BBI’s inhibition of serine protease (trypsin and chymotrypsin) activity. The lack of molecular probes for the isolation of this protein has made it difficult to work with, limiting progress as a significant candidate in the treatment of cancer. This study has successfully identified a set of novel synthetic peptides targeting the BBI, and has demonstrated the ability to bind BBI in vitro. One of those probes has been covalently immobilized on superparamagnetic microbeads to allow the isolation of BBI from soy whey mixtures in a single step. Our ultimate goal is the use of the described synthetic probe to facilitate the isolation of this potential therapeutically protein for low cost, scalable analysis and production of BBI.
User Published Research Sterkel M, Oliveira P, Urlaub H, Harnandez-Martinez S, Rivera-Pomar R, Ons S. OKB, a novel family of brain-gut neuropeptides from insects. Insect Biochemistry and Molecular Biology. 2012 Mar 28. [In Press, Uncorrected Proof].
In insects, neuropeptides play a central role in the control of most physiological processes. The knowledge and characterization of new neuropeptide families, is of interest on the fields of Genetics, Genomics, Neurobiology, Endocrinology and Evolution. This knowledge also provides the tools for the design of peptidomimetics, pseudopeptides or small molecules, capable of disrupting the physiological processes regulated by the signaling molecules and their receptors. This is a promising target for a novel generation of insecticides. Using database searches, mass spectrometry and RACE-PCR, we identified a neuropeptide precursor transcript encoding a new family of insect neuropeptides in the hemipteran Rhodnius prolixus. We named this precursor Orcokinin B, because is originated by the alternative splicing of the Orcokinin gen. EST and genomic data suggests that Orcokinin B is expressed in the nervous system and gut from several insect species, with the exception of Drosophila sp. (Diptera) and Acyirthosiphon pisum (Hemiptera). Mass spectrometry and RT-PCR confirmed the expression of Orcokinin B in brain and anterior midgut of R. prolixus. Furthermore, we identified orthologues of this new family of peptides in genomic and EST databases from Arachnids and Crustaceans.
User Published Research Matt P, VanDuijn M, Brouwer E, Dekker L, Zeneyedpour L, Luider T, Smitt P. Mass spectrometric detection of antigen-specific immunoglobulin peptides in paraneoplastic patient sera. Journal of Autoimmunity. 2012 Mar 27. [In Press, Corrected Proof].
Paraneoplastic neurological syndromes (PNS) are severe immune mediated effects of cancer. The presence of IgG autoantibodies against onconeural antigens in serum is a hallmark of the disease. Multiple paraneoplastic antibodies have been described, including antibodies against HuD, Yo, amphiphysin and CV2. In this study, we test the hypothesis that primary amino-acid structures of the antigen binding part of antibodies from various individuals share common sequences that are specific for each auto-antigen. We selected 60 patients with PNS, associated with antibodies against HuD, Yo, Amp or CV2. Affinity purified IgG was separated using SDS-PAGE and IgG heavy chains were excised, trypsinized and subjected to tandem mass spectrometry. We selected masses that uniquely identified a PNS autoantibody group, and used MS/MS fragmentation spectra to obtain information on peptide sequences. Out of 19,173 unique masses, 28 immunoglobulin-derived peptides were found exclusively in samples from a single autoantibody defined PNS group. Our results confirm that specific peptide structures exist in the antigen binding site of IgG that are shared between individuals harboring autoantibodies against the same onconeural antigen. Thus, the immune response in these patients followed converging paths during the rearrangement, selection and maturation of immunoglobulin sequences. The identified peptides can be applied in the diagnosis of PNS, but these data also indicate that a similar approach in a variety of other diseases involving an immune response would have an appealing outlook.
User Published Research Sugiura T, Mori T, Kamei I, Hirai H, Kawagishi H, and Kondo R. Improvement of ligninolytic properties in the hyper lignin‐degrading fungus Phanerochaete sordida YK‐624 using a novel gene promoter. FEMS Microbiology Letters. 27 March 2012 [E-Published for future editions].
We identified a highly expressed protein (BUNA2) by two-dimensional gel electrophoresis from the hyper lignin-degrading fungus Phanerochaete sordida YK-624 under wood-rotting conditions. Partial amino acid sequences of BUNA2 were determined by LC-MS/MS analysis, and BUNA2 gene (bee2) and promoter region were PCR cloned and sequenced. The bee2 promoter was used to drive expression of the manganese peroxidase gene (mnp4) in P. sordida YK-624. Eighteen mnp4-expressing clones were obtained, with most showing higher ligninolytic activity and selectively than wild-type YK-624. Examination of the ligninolytic properties of the most effective lignin-degrading transformant, BM-65, cultured on wood meal revealed this strain exhibited higher lignin degradation and MnP activities than those of wild type. Transcriptional analysis confirmed the elevated expression of recombinant mnp4 in the transformant. These results indicate that use of the bee2 promoter to drive expression of ligninolytic enzymes may be an effective approach for improving the lignin-degrading properties of white-rot fungi.
User Published Research Xia Y, Bamdad F, Ganzle M, and Chen L. Fractionation and characterization of antioxidant peptides derived from barley glutelin by enzymatic hydrolysis. Food Chemistry. 22 March 2012. [In Press, Accepted Manuscript].
The antioxidant properties of the barley glutelin hydrolysates were evaluated based on their radical scavenging capacity (DPPH/O2·¯/OH˙), Fe2+-chelating effect and reducing power. Alcalase hydrolysates (AH) demonstrated significantly higher antioxidant capacity than those treated by flavourzyme in most of the assays. The AH was separated using ultra-filtration and reversed-phase chromatography, and assessment of the fractions indicated that the large-sized peptides (Mw > 10 kDa) possessed stronger DPPH scavenging activity and reducing power, whereas small-sized peptides (Mw < 1 kDa) were more effective in Fe2+-chelating and OH˙ scavenging effect. The hydrophobic fraction contributed more to Fe2+-chelating and OH˙ scavenging activity. Four peptides contributing to antioxidant activities were identified using LC-MS/MS: Gln-Lys-Pro-Phe-Pro-Gln-Gln-Pro-Pro-Phe, Pro-Gln-Ile-Pro-Glu-Gln-Phe, Leu-Arg-Thr-Leu-Pro-Met and Ser-Val-Asn-Val-Pro-Leu. Compared to the positive controls, AH exhibited excellent Fe2+-chelating activity and strong DPPH/OH˙scavenging effect. Thus hydrolyzed barley glutelin is a potential source of antioxidant peptides for food and nutraceutical applications.
User Published Research Abdelkafia S, Abousalhamc A, Fendrib I, Ogatad H, Barouhe N, Fouquetf B, Scheirlinckxf F, Villeneuvee P, Carrièrea F. Identification of a new phospholipase D in Carica papaya latex. Gene. 2012 Mar 16. [Epub ahead of print].
Phospholipase D (PLD) is a lipolytic enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. It catalyzes the hydrolysis and transphosphatidylation of glycerophospholipids at the terminal phosphodiester bond. The presence of a PLD in the latex of Carica papaya (CpPLD1) was demonstrated by transphosphatidylation of phosphatidylcholine (PtdCho) in the presence of 2% ethanol. Although the protein could not be purified to homogeneity due to its presence in high molecular mass aggregates, a protein band was separated by SDS-PAGE after SDS/chloroform–methanol/TCA–acetone extraction of the latex insoluble fraction. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by micro-LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (723 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 2424 bp encoding a protein of 808 amino acid residues, with a theoretical molecular mass of 92.05 kDa. From sequence analysis, CpPLD1 was identified as a PLD belonging to the plant phosphatidylcholine phosphatidohydrolase family.
User Published Research Gutiérrez-Sánchez G, Atwood J, Kolli VSK, Roussos S, and Augur C. Initial Proteome Analysis of Caffeine-Induced Proteins in Aspergillus tamarii Using Two-Dimensional Fluorescence Difference Gel Electrophoresis. Applied Biochemistry and Biotechnology. 2012 Mar 07. [Epub ahead of print].
Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.
User Published Research Dauly C, Escoubas P, King GF, Nicholson GM. Characterization of Spider Venom Peptides by High-Resolution LC-MS/MS Analysis. Application Note: 511, Thermo Fisher Scientific Inc. 2012.
Animal venoms are natural libraries of biologically active peptides. They encompass a wide variety of structures and pharmacological activities and represent an enormous resource of novel molecules to be used as insecticide, therapeutic and drug models. Australian funnel-web spiders contain many hundreds of peptides that follow a bimodal distribution, with about 75% of the peptides ranging from 3,000 to 5,000 Da1. Although the up-to-date, funnel-web spiders protein database contains about 100 sequences, it was shown recently that the number of peptides present in venom is in the high hundreds1. Venom profiling by high resolution and accurate-mass (HR/AM) LC-MS/MS can therefore be used for species identification and to indicate the presence of potentially unknown toxins. Combining MS-based peptide sequence tags with cutting-edge transcriptomics studies and the ability to generate full peptide sequences using molecular biology techniques such as RACE (Rapid Amplification of Complementary Ends) will allow the generation of large peptide sequence libraries that can be mined for drug discovery purposes, in a global approach termed “venomics”.
User Published Research Robinson MR, Madsen JA, Brodbelt JS. 193 nm Ultraviolet Photodissociation of Imidazolinylated Lys-N Peptides for De Novo Sequencing. Electrophoresis. Anal Chem. 2012 Jan 27.
The goal of many MS/MS de novo sequencing strategies is to generate a single product ion series that can be used to determine the precursor ion sequence. Most methods fall short of achieving such simplified spectra, and the presence of additional ion series impede peptide identification. The present study aims to solve the problem of confounding ion series by enhancing the formation of "golden" sets of a, b, and c ions for sequencing. Taking advantage of the characteristic mass differences between the golden ions allows N-terminal fragments to be readily identified while other ion series are excluded. By combining the use of Lys-N, an alternate protease, to produce peptides with lysine residues at each N-terminus with subsequent imidazolinylation of the -amino group of each lysine, peptides with highly basic sites localized at each N-terminus are generated. Subsequent MS/MS analysis by using 193 nm ultraviolet photodissociation (UVPD) results in enhanced formation of the diagnostic golden pairs and golden triplets that are ideal for de novo sequencing.
User Published Research Giuliano S, Iadarola P, Leva V, Montecucco A, Camerini S, Crescenzi M, Salvini R, Bardoni A. An Insight into the Abundant Proteome of 46BR. 1G1 Fibroblasts Deficient of DNA Ligase I. Electrophoresis. 2012 Jan;33(2):307-15.
This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.
User Published Research Trevors JT, Bej AK, Mojib N, van Elsas JD, Van Overbeek L. Bacterial Gene Expression at Low Temperatures. Extremophiles. 2012 Jan 3.
Under suboptimal environmental conditions such as low temperatures, many bacteria have an extended lag phase, altered cell structures, and composition such as a less fluid (more rigid) and leaky cytoplasmic membrane. As a result, cells may die, enter into a starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. In the latter state, the amount of gene expression per cell is virtually undetectable. In this article, gene expression under (suboptimal) low temperature conditions in non-psychrophilic environmental bacteria is examined. The pros and cons of some of the molecular methodologies for gene expression analysis are also discussed.
User Published Research Eun-Mi K, Juhan K, Joo-Hyun S, Jun-Seong P, Duck-Hee K, and Byung-Gee K. Identification and Characterization of the Rhizobium sp. Strain GIN611 Glycoside Oxidoreductase Resulting in the Deglycosylation of Ginsenosides. Applied and Environmental Microbiology, 2012, 78(1):242-249
Using enrichment culture, Rhizobium sp. strain GIN611 was isolated as having activity for deglycosylation of a ginsenoside, compound K (CK). The purified heterodimeric protein complex from Rhizobium sp. GIN611 consisted of two subunits with molecular masses of 63.5 kDa and 17.5 kDa. In the genome, the coding sequence for the small subunit was located right after the sequence for the large subunit, with one nucleotide overlapping. The large subunit showed CK oxidation activity, and the deglycosylation of compound K was performed via oxidation of ginsenoside glucose by glycoside oxidoreductase. Coexpression of the small subunit helped soluble expression of the large subunit in recombinant Escherichia coli. The purified large subunit also showed oxidation activity against other ginsenoside compounds, such as Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, F1, and the isoflavone daidzin, but at a much lower rate. When oxidized CK was extracted and incubated in phosphate buffer with or without enzyme, (S)-protopanaxadiol [PPD(S)] was detected in both cases, which suggests that deglycosylation of oxidized glucose is spontaneous.
User Published Research Mulema J, Okori P, and Denby K. Proteomic analysis of the Arabidopsis thaliana-Botrytis cinerea interaction using two-dimensional liquid chromatography. African Journal of Biotechnology Vol. 10(76), pp. 17551-17563, 30 November, 2011
A two-dimensional liquid chromatography (2D LC) system, ProteomeLab PF 2D, was employed to study the defence proteome of Arabidopsis following infection with the necrotrophic fungal pathogen, Botrytis cinerea. This system demonstrated differential protein expression in control and treated samples in some fractions. However, the amount of proteins in the fractions and the level to which they were changing could not be established. Among the proteins identified in the fractions that displayed an increase in absorbance was catalase 3 and glutathione S-transferases, demonstrating the importance of an antioxidant system in defence against B. cinerea. Most of the proteins were identified in fractions that displayed reduction in absorbance. Functional categorisation of the identified proteins demonstrated the overrepresentation of photosynthetic pathway, a phenomenon also observed in other host and nonhost pathogen interactions. Proteomelab PF 2D did not identify as many proteins as expected possibly due to the masking effect of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) as this protein was identified in almost all fractions including those having an increase in absorbance. Depletion of this protein from crude plant protein extracts is likely to improve protein identification by mass spectrometry, especially for the low abundant proteins. A number of proteins were identified in each fraction and it was difficult to discern which of the proteins was responsible for the differential increase or reduction in absorbance.
User Published Research Kumar SM, Selvam K. Isolation and Purification of High Efficiency L-asparaginase by Quantitative Preparative Continuous-elution SDS PAGE Electrophoresis. J Microbial Biochem Technol 2011, 3:5. 2011 Dec 4.
An Unique extracelluar glutaminase free L-asparaginase from novel marine Actinomycetes was isolated to perceptible homogeneity in agro industrial wastes. Quantitative Preparative Continuous-Elution SDS PAGE Electrophoresis is a high-resolution method for the preparative isolation of L-Asparaginase in biological samples. The enzyme was purified 248.68-fold and showed a final specific activity of 5035.28 IU/mg with an 80.71% yield. The homotetramer enzyme has a molecular mass of 133.25 kDa and an isoelectric point of approximately 5.4.Kinetic parameters, Km and Vmax of purified L-asparaginase from Streptomyces radiopugnans MS1 were found to be 0.0598, 3.5478 IU μg-1 respectively. The de novo sequencing strategy presented here provides a rapid and reliable means to identify proteins in Streptomyces radiopugnans MS1. The purified L-asparaginase has no glutaminase activity, which can diminish the leeway of side effects during the itinerary of anti-malignancy therapy.
User Published Research Singh S, Braus-Stromeyer SA, Timpner C, Valerius O, von Tiedemann A, Karlovsky P, Druebert C, Polle A, Braus GH. The plant host Brassica napus induces in the pathogen Verticillium longisporum the expression of functional catalase peroxidase which is required for the late phase of disease. Mol Plant Microbe Interact. 2011 Nov 23.
The devastating soil-borne fungal pathogen Verticillium longisporum is host-specific to Brassicaceae including oilseed rape (Brassica napus) as the economically most important crop. The fungus infects through the roots and causes stunting and early senescence of susceptible host plants and a marked decrease in crop yield. We show here that V. longisporum reacts to the presence of B. napus xylem sap with the production of six distinct up-regulated and eight down-regulated proteins visualized by 2-dimensional gel electrophoresis. Identification of ten proteins by mass spectrometry revealed that all up-regulated proteins are involved in oxidative stress response. The catalase peroxidase VlcpeA was the most up-regulated protein and is encoded by two isogenes, VlcpeA-1 and VlcpeA-2. Both genes are 98% identical corroborating the diploid or 'amphihaploid' status of the fungus. Knock-downs of both VlcpeA genes reduced protein expression by 80% and resulted in sensitivity against reactive oxygen species. Whereas saprophytic growth and the initial phase of the plant infection were phenotypically unaffected, the mutants were not able to perform the late phases of disease. We propose that the catalase peroxidase plays a role in protecting the fungus from the oxidative stress generated by the host plant at an advanced phase of the disease.
User Published Research Broodman I, de Costa D, Stingl C, Dekker LJ, Vanduijn MM, Lindemans J, van Klaveren RJ, Luider TM. Mass spectrometry analyses of kappa and lambda fractions result in increased number of complementarity determining regions identifications. Proteomics. 2011 Nov 25.
Sera from lung cancer patients contain antibodies against tumor associated antigens. Specific amino acid sequences of the complementarity determining regions (CDRs) in the antigen-binding fragment (Fab) of these antibodies have potential as lung cancer biomarkers. Detection and identification of CDRs by mass spectrometry can significantly be improved by reduction of the complexity of the immunoglobulin molecule. Our aim was to molecular dissect IgG into kappa and lambda fragments to reduce the complexity and thereby identify substantially more CDRs than by just total Fab isolation. We purified Fab, Fab-κ, Fab-λ, κ and λ light chains from serum from 10 stage I lung adenocarcinoma patients and 10 matched controls from current and former smokers. After purification, the immunoglobulin fragments were enzymatically digested and measured by high-resolution mass spectrometry. Finally, we compared the number of CDRs identified in these immunoglobulin fragments with that in the Fab fragments. Twice as many CDRs were identified when Fab-κ, Fab-λ, ê and λ (3330) were combined than in the Fab fraction (1663) alone. The number of CDRs and κ:λ ratio was statistically similar in both cases and controls. Molecular dissection of IgG identifies significantly more CDRs, which increases the likelihood of finding lung cancer-related CDR sequences.
User Published Research Labbate M, Boucher Y, Roy Chowdhury P, Stokes HW. Integration of a laterally acquired gene into a cell network important for growth in a strain of Vibrio rotiferianus. BMC Microbiology. 2011 Nov 18.
Background: Lateral Gene Transfer (LGT) is a major contributor to bacterial evolution and up to 25% of a bacterium's genome may have been acquired by this process over evolutionary periods of time. Successful LGT requires both the physical transfer of DNA and its successful incorporation into the host cell. One system that contributes to this latter step by site-specific recombination is the integron. Integrons are found in many diverse bacterial Genera and is a genetic system ubiquitous in vibrios that captures mobile DNA at a dedicated site. The presence of integron-associated genes, contained within units of mobile DNA called gene cassettes makes up a substantial component of the vibrio genome (1-3%). Little is known about the role of this system since the vast majority of genes in vibrio arrays are highly novel and functions cannot be ascribed. It is generally regarded that strain-specific mobile genes cannot be readily integrated into the cellular machinery since any perturbation of core metabolism is likely to result in a loss of fitness.
Results: In this study, at least one mobile gene contained within the Vibrio rotiferianus strain DAT722, but lacking close relatives elsewhere, is shown to greatly reduce host fitness when deleted and tested in growth assays. The precise role of the mobile gene product is unknown but impacts on the regulation of outermembrane porins. This demonstrates that strain specific laterally acquired mobile DNA can be integrated rapidly into bacterial networks such that it becomes advantageous for survival and adaptation in changing environments.
Conclusions: Mobile genes that are highly strain specific are generally believed to act in isolation. This is because perturbation of existing cell machinery by the acquisition of a new gene by LGT is highly likely to lower fitness. In contrast, we show here that at least one mobile gene, apparently unique to a strain, encodes a product that has integrated into central cellular metabolic processes such that it greatly lowers fitness when lost under those conditions likely to be commonly encountered for the free living cell. This has ramifications for our understanding of the role mobile gene encoded products play in the cell from a systems biology perspective.
User Published Research Révay T, Villagómez DA, Brewer D, Chenier T, King WA. GTG Mutation in the Start Codon of the Androgen Receptor Gene in a Family of Horses with 64,XY Disorder of Sex Development. Sexual Development. 2011 Nov 15.
Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRY(+) disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.
User Published Research Jeon AH, Schmitt-Ulms G. Time-controlled transcardiac perfusion crosslinking for in vivo interactome studies. Methods Mol Biol. 2012;803:231-46.
The time-controlled transcardiac perfusion crosslinking (tcTPC) method differs from conventional perfusion fixation in that the crosslinking reagent is administered throughout the circulatory system for only a relatively short period of time, thereby allowing limited crosslinking to occur. Bait protein complexes are isolated by affinity capture (AC) under stringent conditions and are recovered from the AC matrix by acidic elution. Affinity-purified proteins are reduced, alkylated, and digested with a specific endoproteinase, such as trypsin. Subsequently, peptides are isotopically labeled, separated by reversed-phase chromatography and analyzed by quantitative tandem mass spectrometry (MS/MS). The proteins crosslinked to the bait protein during tcTPC are identified by database searches with conventional protein identification software. The tcTPC strategy offers unique advantages over alternative approaches for studying a subset of protein complexes which require a particular environment for their structural integrity, such as membrane protein complexes that are notorious for their tendency to dissociate upon detergent solubilization. The sensitivity and utility of this method are influenced by the spatial distribution of chemical groups within the bait protein complexes that can engage in productive crosslinks.
User Published Research Cheng Z, Woody OZ, McConkey BJ, Glick BR. Combined effects of the plant growth-promoting bacterium Pseudomonas putida UW4 and salinity stress on the Brassica napus proteome. Applied Soil Ecology. 2011 November 15.
The influences of two strains of Pseudomonas putida UW4, a plant growth-promoting bacterium (PGPB) on the shoot and root proteomic profiles of Brassica napus (canola) were examined under salinity stress using two-dimensional difference gel electrophoresis. The two strains (wild-type UW4 and an AcdS minus mutant strain) differ in the availability of a functional 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, a bacterial protein known to play a role in host plant's stress responses. In total, ninety protein spots (representing 76 different proteins) with significantly altered expression levels in the presence of salt and/or bacteria were identified by mass spectrometry. Many of the identified proteins are involved in photosynthesis, anti-oxidative processes, transportation across membranes, and pathogenesis-related responses. The effects of salt and bacteria on the canola proteome were shown to be quite diverse, with salinity stress causing more dramatic plant protein expression changes than bacteria. In addition, bacteria were demonstrated to moderate some of the salt effects on plant protein differential expression. This work contributes to the understanding of how plant protein expression is affected by various environmental signals.
User Published Research Petruzziello F, Fouillen L, Wadensten H, Kretz R, Andren PE, Rainer G, Zhang X. Extensive Characterization of Tupaia Belangeri Neuropeptidome Using an Integrated Mass Spectrometric Approach.. Journal of Proteome Research. 2011 November 9.
Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed and targeted liquid chromatography (LC)-Fourier Transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have identical or similar sequences to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.
User Published Research Darvillea LNF, Merchantb ME, Macchab V, Siddavarapub VR, Hasana V, Murraya KK. Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator Mississippiensis). Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2011 November 9.
Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.
User Published Research Lindop R, Arentz G, Chataway TK, Thurgood LA, Jackson MW, Reed JH, McCluskey J, Gordon TP. Molecular Signature of a Public Clonotypic Autoantibody in Primary Sjögren's Syndrome: A “Forbidden” Clone in Systemic Autoimmunity. Arthritis & Rheumatism, 63: 3477–3486. 2011 October 28.
Objective: This study was undertaken to determine the molecular characteristics of clonotypic autoantibodies in the sera of patients with primary Sjögren's syndrome (SS). This characterization is hampered by the presence of mixed anti-Ro/La specificities that may conceal clonotypic species. In order to narrow clonotypic diversity, a positive selection step was performed on a peg-like determinant of Ro 60 (termed Ro 60–peg) prior to analysis of the autoantibody proteome.
Methods: Monospecific anti–Ro 60–peg IgG were isolated by affinity purification from the sera of 7 patients with primary SS and anti-Ro/La and subjected to 2-dimensional gel electrophoresis and high-resolution orbitrap mass spectrometric sequencing. V regions of heavy and light chains were analyzed by combined database and de novo amino acid sequencing.
Results: Proteomic analysis revealed a Ro 60–peg–specific IgG1κ-restricted monoclonal autoantibody that was present in the sera of all patients and specified by a VH3–23 heavy chain paired with a Vκ3–20 light chain. The public anti–Ro 60–peg clonotype was specified further by common mutations in the heavy-chain and light-chain complementarity-determining regions. Titers and relative affinities of clonotypic IgG did not vary over the course of the disease.
Conclusion: The expression of a Ro 60–reactive public B cell clonotype in a subset of patients with primary SS as a long-lived, class-switched circulating autoantibody implies a common breach of B cell tolerance checkpoints in these patients. The unique heavy chain/light chain signature opens the possibility of tracking the development of a “forbidden” clone against a bona fide systemic autoantigen in human disease.
User Published Research DiFonzo A, Soffientini P, Manousidou T. Evaluation of Two Separation Techniques, SCX and OFFGEL and of Two Fragmentation Methods, CAD and PQD, to Asses iTRAQ Quantitation Efficiency. Journal of Biomolecular Techniques. 22(Supplement): S67; 2011 October 25.
To evaluate various sample preparation and analysis platforms for isobaric tags for iTRAQ experiments, we compared two separation and two fragmentation techniques: Strong Cation Exchange (SCX) with isoelectric point separation. In addition, we compared two types of mass analyzers: a quadrupole and a linear ion trap, the former within a quad-TOF hybrid mass spectrometer, and the later within an LTQ-FT hybrid MS. This was done by comparing two fragmentation techniques, collisionally activated dissociation (CAD) with pulsed Q dissociation (PQD). Proteins from lysed HeLa cells were trypsinized and peptides were iTRAQ labeled. Half of the labeled digest was separated via SCX into20 fractions. The other half of digested peptides was separated with an OFFGEL isoelectric point fractionator into 24 fractions. Each dried peptide fraction was desalted, and submitted to reverse-phase C-18 HPLC on-line with the mass spectrometer. Quantitative data analysis for the LTQ-FT data was performed using Proteome Discoverer (Thermo Electron) and for the QStar data, PEAKS Studio (Bioinformatic Solution Inc.). R software was used for the statistical validation of results. The LTQ-FT data was also analyzed with the PEAKS software to asses the viability of PEAKS as a universally applicable data analysis package. Comparison between separation and dissociation was based on a) number of proteins quantified b) reproducibility of technical replicates based on protein distribution c) standard deviation for data obtained from the two dissociation techniques. Fractionation with OFFGEL increased the number of identified and quantified of proteins by approximately 20%. Number of mass spectra which can be used for quantitation is higher from the QStar instrument implying that the order of magnitude increased sensitivity of the LTQ-FT relative to the quad-TOF is lost to the inefficiency of the PQD. Reproducibility of technical replicates from PQD is lower than that obtained with data from CAD.
User Published Research Smeulders MJ, Barends TRM, Pol A, Scherer A, Zandvoort MH, Udvarhelyi A, Khadem AF, Menzel A, Hermans J, Shoeman RL, Wessels HJCT, van den Heuvel LP, Russ L, Schlichting I, Jetten MSM, Op den Camp HJM. Evolution of a New Enzyme for Carbon Disulphide Conversion by an Acidothermophilic Archaeon. Nature. 2011 October 19.
Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H2S) and carbon disulphide (CS2)1, 2. The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS2 into H2S and carbon dioxide (CO2), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS2 hydrolase from Acidianus A1-3. The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO2 is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme’s substrate specificity for CS2, which is hydrophobic. The transposon sequences that surround the gene encoding this CS2 hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient β-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS2 metabolism.
User Published Research Miller L, Hou X, Gagnidze1 K, Sweedler JV, Rodriguiz RM, Devi1 LA, Wetsel WC. Mice Deficient in Endothelin-Converting Enzyme-2 Exhibit Abnormal Responses to Morphine and Altered Peptide Levels in the Spinal Cord. Journal of Neurochemistry. 2011 Oct 11.
An increasing body of evidence suggests that Endothelin-converting enzyme-2 (ECE-2) is a non-classical neuropeptide processing enzyme. Similar to other neuropeptide processing enzymes, ECE-2 exhibits restricted neuroendocrine distribution, intracellular localization, and an acidic pH optimum. However, unlike classical neuropeptide processing enzymes, ECE-2 exhibits a non-classical cleavage site preference for aliphatic and aromatic residues. We previously reported that ECE-2 cleaves a number of neuropeptides at non-classical sites in vitro; however its role in peptide processing in vivo is poorly understood. Given the recognized roles of neuropeptides in pain and opiate responses, we hypothesized that ECE-2 knockout (KO) mice might show altered pain and morphine responses compared to wild-type (WT) mice. We find that ECE-2 KO mice show decreased response to a single injection of morphine in hot-plate and tail-flick tests. ECE-2 KO mice also show more rapid development of tolerance with prolonged morphine treatment and fewer signs of naloxone-precipitated withdrawal. Peptidomic analyses revealed changes in the levels of a number of spinal cord peptides in ECE-2 KO as compared to WT mice. Taken together, our findings suggest a role for ECE-2 in the non-classical processing of spinal cord peptides and morphine responses; however, the precise mechanisms through which ECE-2 influences morphine tolerance and withdrawal remain unclear.
User Published Research Kim DR, Gidvani RD, Ingalls BP, Duncker BP, McConkey BJ. Differential Chromatin Proteomics of the MMS-induced DNA Damage Response in Yeast. Proteome Science. October 2011; 9:62
Background: Protein enrichment by sub-cellular fractionation was combined with differentialin-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins.
Results: Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress.
Conclusion: The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response.
User Published Research Yin P, Hou X, Romanova EV, Sweedler JV. Neuropeptidomics: Mass Spectrometry-Based Qualitative and Quantitative Analysis. Methods Mol Biol. 2011;789:223-36.
Neuropeptidomics refers to a global characterization approach for the investigation of neuropeptides, often under specific physiological conditions. Neuropeptides comprise a complex set of signaling molecules that are involved in regulatory functions and behavioral control in the nervous system. Neuropeptidomics is inherently challenging because neuropeptides are spatially, temporally, and chemically heterogeneous, making them difficult to predict in silico from genomic information. Mature neuropeptides are produced from intricate enzymatic processing of precursor proteins/prohormones via a range of posttranslational modifications, resulting in multiple final peptide products from each prohormone gene. Although there are several methods for targeted peptide studies, mass spectrometry (MS), with its qualitative and quantitative capabilities, is ideally suited to the task. MS provides fast, sensitive, accurate, and high-throughput peptidomic analysis of neuropeptides without requiring prior knowledge of the peptide sequences. Aided by liquid chromatography (LC) separations and bioinformatics, MS is quickly becoming a leading technique in neuropeptidomics. This chapter describes several LC-MS analytical methods to identify, characterize, and quantify neuropeptides while emphasizing the sample preparation steps so integral to experimental success.
User Poster Research Rykl J. Identification of Degradation Products of Synthetic Peptides with Nano-LC/MS on an Orbitrap Mass Spectrometer. HUPO 2011: P1605.
Purpose: Synthetic peptides are widely used as therapeutic drugs. In order to guarantee their biological activity, they must be characterized thoroughly. Also, during long-term storage, degradation of the product may occur.
Methods: The peptides calcitonin and glucagon were stored long-term under different conditions and sub-sequently analyzed using an Orbitrap mass spectrometer coupled to a nano-LC system.
Results: Degradation of synthetic peptides can result in complex mixtures. Even low amounts of degradation products can be identified and quantified using nano-LC-coupled mass spectrometry (MS).
User Published Research Nowell V, Prescott JF. Genomic and Proteomic Analysis of a Bovine Hemorrhagic Abomasitis Type a Clostridium Perfringens Isolate. Nowell. 2011 September.
This study sought to understand the pathogenesis of hemorrhagic abomasitis in calves by characterizing a type A Clostridium perfringens isolate. The complete genome sequence of an isolate from an outbreak of hemorrhagic abomasitis was compared to the three complete C. perfringens genomes currently available in GenBank. Unique findings included the presence of an integrated plasmid sequence and a frameshift mutation in the virS gene, which encodes the main sensor kinase that controls virulence gene regulation. An ~ 55 kb plasmid similar to pCW3 was found, in addition to two smaller plasmids with no significant similarity to available C. perfringens plasmid sequences. A number of plasmid-related fragments were also identified. Neither genomic nor proteomic approaches identified novel toxins, but an alternate and unexpected picture of virulence has emerged suggesting that anomalous virulence gene regulation might contribute to pathogenicity in this isolate.
User Published Research Yu J, Chen S, Zhao Q, Wang T, Yang C, Diaz C, Sun G, Dai S. Physiological and Proteomic Analysis of Salinity Tolerance in Puccinellia tenuiflora. Journal of Proteome Research. 2011 Sep 2;10(9):3852-70.
Soil salinity poses a serious threat to agriculture productivity throughout the world. Studying mechanisms of salinity tolerance in halophytic plants will provide valuable information for engineering plants for enhanced salt tolerance. Monocotyledonous Puccinellia tenuiflora is a halophytic species that widely distributed in the saline-alkali soil of the Songnen plain in northeastern China. Here we investigate the molecular mechanisms underlying moderate salt tolerance of P. tenuiflora using a combined physiological and proteomic approach. The changes in biomass, inorganic ion content, osmolytes, photosynthesis, defense-related enzyme activities, and metabolites in the course of salt treatment were analyzed in the leaves. Comparative proteomic analysis revealed 107 identities (representing 93 unique proteins) differentially expressed in P. tenuiflora leaves under saline conditions. These proteins were mainly involved in photosynthesis, stress and defense, carbohydrate and energy metabolism, protein metabolism, signaling, membrane, and transport. Our results showed that reduction of photosynthesis under salt treatment was attributed to the down-regulation of the light-harvesting complex (LHC) and Calvin cycle enzymes. Selective uptake of inorganic ions, high K(+)/Na(+) ratio, Ca(2+) concentration changes, and an accumulation of osmolytes contributed to ion balance and osmotic adjustment in leaf cells. Importantly, P. tenuiflora plants developed diverse reactive oxygen species (ROS) scavenging mechanisms in their leaves to cope with moderate salinity, including enhancement of the photorespiration pathway and thermal dissipation, synthesis of the low-molecular-weight antioxidant α-tocopherol, and an accumulation of compatible solutes. This study provides important information toward improving salt tolerance of cereals.
User Published Research Asoodeh A, Yazdi MM, Chamani JK. Purification and Characterisation of Angiotensin I Converting Enzyme Inhibitory Peptides from Lysozyme Hydrolysates. Food Chemistry. 2011 August 22.
Hen egg white lysozyme (HEWL) was hydrolysed with trypsin, papain and a combination of the two. The prepared hydrolysates exhibited ACE inhibitory activity. The hydrolysates were fractionated using ultrafiltration and reverse phase-high performance liquid chromatography (RP-HPLC). Three fractions, which showed the highest ACE inhibitory activities, were purified by RP-HPLC. They were the F7 (from papain–trypsin hydrolysate), F8 (from papain hydrolysate) and F3 (from trypsin hydrolysate) fractions. The IC50 values were 0.03, 0.155 and 0.23 mg/ml for F7, F8 and F3, respectively. The F7 fraction was the most potent ACE inhibitor peptide, and was composed of 12 amino acids, Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-Arg (MW: 1428.6 Da). Lineweaver–Burk plots suggest that the F7 peptide acts as an uncompetitive inhibitor against ACE. The kinetic parameters (Km, Vmax, and Ki) for the F7 peptide were measured and compared to the control.
User Published Research Oliveira LMA, Lages A, Gomes RA, Neves H, Familia C, Coelho AV, Quintas A. Insulin Glycation by Methylglyoxal Results in Native-like Aggregation and Inhibition of Fibril Formation. BMC Biochemistry, 12:41. 2011 August 5.
Background: Insulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.
Results: Methylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.
Conclusions: We propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-beta core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.
User Published Research Thomas R, Twine SM, Fulton KM, Tessier L, Kilmury SLN, Ding W, Harmer N, Michell SL, Oyston PCF, Titball RW, Prior JL. Glycosylation of DsbA in Francisella Tularensis Subspecies Tularensis. Journal of Bacteriology. 2011 August 4.
In Francisella tularensis subspecies tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots in two dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1156 Da glycan moiety in O-linkage. Mass spectrometry studies suggest the glycan is a hexasaccharide, comprised of N-acetyl hexosamines, hexoses and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798) resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis FTT0798 and FTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis.
User Published Research Boerjan B, Vandingenena K, De Loofa A, Schoofsa L. In Search for Non-Steroidogenic Functions of the Prothoracic Glands of the Desert Locust, Schistocerca Gregaria: A Peptidomic and Proteomic Approach. Peptides. 2011 July 24.
The only well established function of the prothoracic glands (PGs) of insects is the production of ecdysteroids. In gregarious locusts, like in most insect species, the PGs degenerate soon after the adult moult. In this way they resemble the thymus of mammals, a gland with an important role in the build up of the immune system in young animals. In adult solitarious locusts the PGs persist much longer, however without producing substantial amounts of ecdysteroids. In the literature the existence of a well developed rough endoplasmic reticulum and Golgi complex system has been repeatedly reported, suggesting an active role in peptide or/and protein synthesis and release. The nature of the secreted products remained unknown. Our pepdidomic analysis of an acidified methanolic extract of PGs of last instar gregarious nymphs did not yield any indication for the presence of known locust or other peptides. The peptide release assay was also negative. For our proteomic analysis, we developed an EST-based identification strategy. We successfully identified 50 protein spots on a two dimensional map. In addition to typical protein synthesis-related proteins, a number of proteins with a role in detoxification processes were found, suggesting some role of the PGs in the defence system.
User Published Research Sterkel M, Urlaub H, Rivera-Pomar R, Ons S. Functional Proteomics of Neuropeptidome Dynamics During the Feeding Process of Rhodnius Prolixus. Journal of Proteome Research. 2011 June 24.
In hematophagous insects, blood intake triggers a prompt response mediated by neuropeptides, which regulates a variety of physiological processes. Here we report a quantitative proteomic analysis of the post-feeding response in the central nervous system of Rhodnius prolixus, a vector of Chagas disease. The concentration of neuropeptides NVP-like, ITG-like, kinin-precursor peptide and neuropeptide-like precursor 1 (NPLP1) significantly changes in response to blood intake. We also performed a neuropeptidomic analysis of other feeding-related organs, namely salivary glands and gut. We identified NPLP1 in salivary glands and myosuppressin in midgut. This is the first report suggesting a role for NPLP1, involving the peptides processed from this precursor in the hormonal control of the production and/or release of saliva. Our results contribute to the understanding of the post-prandial neuroendocrine response in hematophagous and provide important information for physiological and pharmacological studies aimed to the design of next-generation insecticides such as peptidomimetics.
User Published Research Hamritaa B, Nasra HB, Hammann P, Kuhnb L, Guillierc CL, Chaieba A, Khairia H, Chaheda K. An Elongation Factor-Like protein (EF-Tu) Elicits a Humoral Response in Infiltrating Ductal Breast Carcinomas: An Immunoproteomics Investigation. BMC Genomics;12(1):268. 2011 May 26.
Objectives:In the current study, we have used an immunoproteomics approach to identify proteins that commonly elicit a humoral response in patients with infiltrating ductal carcinomas of the breast.
Design and methods: Sera obtained at the time of diagnosis from 40 patients with invasive breast cancer and 42 healthy controls were screened for the presence of IgG antibodies to MCF-7 cell line proteins using a serological proteomics-based approach.
Results: An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu.
Conclusions:The immunoproteomic approach implemented here offers a powerful tool for determining novel tumor antigens that induce a humoral immune response in cancer patients. From our findings, the immunoreactive EF-Tu protein and/or the related circulating antibodies may display clinical usefulness as potential diagnostic markers and provide a means for a better understanding of the molecular mechanisms underlying breast cancer development.
User Published Research Yin F, Pajak A, Chapman R, Sharpe A, Huang S, Marsolais F. Analysis of Common Bean Expressed Sequence Tags Identifies Sulfur Metabolic Pathways Active in Seed and Sulfur-Rich Proteins Highly Expressed in the Absence of Phaseolin and Major Lectins. BMC Genomics;12(1):268. 2011 May 26.
Background: A deficiency in phaseolin and phytohemagglutinin is associated with a near doubling of sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris), particularly cysteine, elevated by 70%, and methionine, elevated by 10%. This mostly takes place at the expense of an abundant non-protein amino acid, S-methyl-cysteine. The deficiency in phaseolin and phytohemagglutinin storage proteins is mainly compensated by increased levels of the 11S globulin legumin and residual lectins. Legumin, albumin-2, defensin and albumin-1 were previously identified as contributing to the increased sulfur amino acid content in the mutant line, on the basis of similarity to proteins from other legumes.
Results: Profiling of free amino acids in developing seeds of the BAT93 reference genotype revealed a biphasic accumulation of gamma-glutamyl-S-methyl-cysteine, the main soluble form of S-methyl-cysteine, with a lag phase occurring during storage protein accumulation. A collection of 30,147 expressed sequence tags (ESTs) was generated from four developmental stages, corresponding to distinct phases of gamma-glutamyl-S-methyl-cysteine accumulation, and covering the transitions to reserve accumulation and dessication. Analysis of gene ontology categories indicated the occurrence of multiple sulfur metabolic pathways, including all enzymatic activities responsible for sulfate assimilation, de novo cysteine and methionine biosynthesis. Integration of genomic and proteomic data enabled the identification and isolation of cDNAs coding for legumin, albumin-2, defensin D1 and albumin-1A and -B induced in the absence of phaseolin and phytohemagglutinin. Their deduced amino acid sequences have a higher content of cysteine than methionine, providing an explanation for the preferential increase of cysteine in the mutant line.
Conclusion: The EST collection provides a foundation to further investigate sulfur metabolism and the differential accumulation of sulfur amino acids in seed of common bean. Identification of sulfur-rich proteins whose levels are elevated in seed lacking phaseolin and phytohemagglutinin and sulfur metabolic genes may assist the improvement of protein quality.
User Published Research Xie F, Romanova EV, Sweedler JV. Neuropeptidomics of the Mammalian Brain. Neuroproteomics Neuromethods, 2011, Volume 57, Part 4. 2011 May 25.
A suite of bioactive peptides orchestrates a variety of cellular interactions in the mammalian brain. A new bioanalytical strategy, neuropeptidomics, has evolved from the quest to characterize these important signaling peptides (SPs). The goal of a neuropeptidomics experiment is to characterize the peptides present in an intact brain, brain region, or individual neuron. To succeed, a neuropeptidomics measurement needs to deal with the large dynamic range and low abundance of some neuropeptides in a background of peptides from postmortem degradation of ubiquitous proteins. Core components of a successful ­neuropeptidomics study include effective tissue sampling, sensitive and robust peptide characterization, and comprehensive data analysis and interpretation. Mass spectrometry (MS) has become the central analytical approach for high-throughput, high-confidence characterization of the brain peptidome because of its capability to detect, identify, and quantify known and unknown peptides. Robust fractionation techniques, such as two-dimensional liquid chromatography, are commonly used in conjunction with MS to enhance investigation of the peptidome. Identification and characterization of peptides are more complex when neuropeptide prohormone genes have not been annotated. This chapter outlines techniques and describes protocols for three different experimental designs that combine MS with liquid chromatography, each aimed at high-throughput discovery of peptides in brain tissue. Further, we describe the currently available bioinformatics tools for automatic query of the experimental data against existing protein databases, as well as manual retrieval of structural information from raw MS data.
User Published Research Rebelloa KM, Barrosoa JSL, Motab EM, Carvalhoa PC, Peralesa J, Lenzib HL, Neves-Ferreiraa AGC. Comprehensive proteomic profiling of adult Angiostrongylus costaricensis, a human parasitic nematode. Journal of Proteomics. 2011 May 16.
Angiostrongylus costaricensis is a nematode helminth that causes an intestinal acute inflammatory process known as abdominal angiostrongyliasis, which is a poorly understood human disease occurring in Latin America. Our aim was to study the proteomic profiles of adult parasites focusing on immunogenic proteins. Total cellular extracts from both genders showed similar 2-DE profiles, with 60% of all protein spots focused between pH 5–7 and presenting molecular masses from 20.1 to 66 kDa. A total of 53 different dominant proteins were identified in our dataset and were mainly associated with the following over-represented Gene Ontology Biological Process terms: “macromolecule metabolic process”, “developmental process”, “response to stress”, and “biological regulation”. Female and male immunoblots showed similar patterns of reactive proteins. Immunoreactive spots identified by MALDI-PSD were found to represent heat shock proteins, a putative abnormal DAuer Formation family member, and galectins. To date, very few biochemical analyses have focused on the nematode Angiostrongylus costaricensis. As such, our results contribute to a better understanding of its biology and the mechanisms underlying the host-parasite relationship associated with this species. Moreover, our findings represent a first step in the search for candidate proteins for diagnostic assays and the treatment of this parasitic infection.
User Published Research Chambala B, Bergenståhlb B, Dejmekb P. Edible Proteins from Coconut Milk Press Cake; One Step Alkaline Extraction and Characterization by Electrophoresis and Mass Spectrometry. Food Research International. 2011 May 7.
Coconut milk press cake, the spent endosperm left over after the extraction of coconut milk, was utilized to extract additional protein under alkaline conditions. The maximum protein yield was observed at 50 C and pH 11 and corresponded to total endosperm protein yield of 71%, favourably comparable to yields of more complex extractions. The extract was characterized by amino acid analysis and 1D SDS-PAGE, 2D electrophoresis and MS-MS spectroscopy. Coconut milk and insoluble milk sediment from the same individual nuts were also analyzed.
The alkaline extract reproducibly differed from the other fractions and from those reported by others in amino acid content as seen in principal component analysis, and had in particular higher threonine content The peptide found in the alkaline extract but with no counterpart in coconut milk or its sediment showed IP (≈ 5) and mass (≈ 40 kDa) and matched partially glutelin from oilpalm.
User Published Research Chaveza JD, Wua J, Bissonb W, Maiera CS. Site-specific proteomic analysis of lipoxidation adducts in cardiac mitochondria reveals chemical diversity of 2-alkenal adduction. Journal of Proteomics. 2011 April 19.
Identification of endogenous protein targets of electrophilic 2-alkenals in cardiac mitochondria; ► An aldehyde/keto-specific chemical labeling and affinity strategy in combination with LC-MS/MS reveals chemical diversity of 2-alkenal adductions; ► Target sites were modified by a variety of 2-alkenal products including acrolein, β-hydroxyacrolein, crotonaldehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal.
User Published Research Robinson MW, Corvo I, Jones PM, George AM, Padula MP, To J, Cancela M, Rinaldi G, Tort JF, Roche L, Dalton JP. Collagenolytic Activities of the Major Secreted Cathepsin L Peptidases Involved in the Virulence of the Helminth Pathogen, Fasciola hepatica. PLoS Negl Trop Dis 5(4). 2011 April 5.
Fasciola hepatica is a helminth parasite that causes liver fluke disease (fasciolosis) in domestic animals (sheep and cattle) and humans worldwide. In order to infect their mammalian hosts, F. hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum and penetrate the liver. After migrating through the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and feed on host haemoglobin to support the production of eggs. To achieve these tasks, F. hepatica secretes a number of distinct cathepsin L cysteine peptidases (FhCL). Thus, the infective larvae that penetrate the host gut secrete cathepsin L3 (FhCL3), the migrating liver-stage juvenile parasites secrete both FhCL1 and FhCL2 while mature bile duct parasites that feed on host blood secrete predominantly FhCL1 but also FhCL2. Here we show that the major cathepsin L peptidases secreted by F. hepatica (FhCL1, FhCL2 and FhCL3) display differential ability to degrade host collagen (an important component of host tissues) and investigate this phenomenon at the molecular level.
User Published Research Farinha AP, Irar S, de Oliveira E, Oliveira M, Pagès M. Novel clues on abiotic stress tolerance emerge from embryo proteome analyses of rice varieties with contrasting stress adaptation. Proteomics. 2011 March 31.
Cereal embryos sustain severe water-deficit at the final stage of seed maturation. The molecular mechanisms underlying the acquisition of desiccation tolerance in seed embryos are similar to those displayed during water-deficit in vegetative tissues. The genetic variation among six rice genotypes adapted to diverse environmental conditions was analysed at the proteome level to get further clues on the mechanisms leading to water-stress tolerance. Mass spectrometry analysis allowed the identification of 28 proteins involved in stress tolerance (LEAs - late embryogenesis abundant proteins), nutrient reservoir activity, among other proteins implicated in diverse cellular processes potentially related to the stress response (e.g., mitochondrial import translocase). Hierarchical clustering and multidimensional scaling analyses revealed a close relationship between the stress-sensitive genotypes, whereas the stress-tolerant varieties were more distantly related. Besides qualitative and significant quantitative changes in embryo proteins across the distinct varieties, we also found differences at post-translational level. The results indicated that LEA Rab21 was more strongly phosphorylated in the embryos of the sensitive varieties than in the embryos of the tolerant ones. We propose that the differences found in the phosphorylation status of Rab21 are related to stress tolerance.
User Published Research Knief C, Delmotte N, Vorholt J. Bacterial adaptation to live in association with plants - a proteomic perspective from culture to in situ conditions. J Proteome Res. 2011 Mar 22.
Diverse bacterial taxa that live in association with plants affect plant health and development. This is most evident for those bacteria that undergo a symbiotic association with plants or infect the plants as pathogens. Proteome analyses have contributed significantly towards a deeper understanding of the molecular mechanisms underlying the development of these associations. They were applied to obtain a general overview of the protein composition of these bacteria, but more so to study effects of plant signaling molecules on the cytosolic proteome composition or metabolic adaptations upon plant colonization. Proteomic analyses are particular useful for the identification of secreted proteins, which are indispensible to manipulate a host plant. Recent advances in the field of proteome analyses have initiated a new research area, the analysis of more complex microbial communities. Such studies are just at its beginning but hold great potential for the future to elucidate not only the interaction between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa when living in association with plants. These include not only the symbiotic and pathogenic bacteria, but also the commensal bacteria that are consistently found in association with plants and whose functions remain currently largely uncovered.
User Published Research Coumans JV, Harvey J, Backhouse D, Poljak A, Raftery MJ, Nehl D, Katz ME, Pereg L. Proteomic assessment of host-associated microevolution in the fungus Thielaviopsis basicola. Environ Microbiol. 2011 Mar;13(3):576-588.
Thielaviopsis basicola, a soil-borne pathogen with a broad host range and a cosmopolitan distribution, is emerging as a major risk to sustainable cotton production in Australia. Previous studies suggested that host specialization has occurred making T. basicola an ideal model for a comparative proteomic analysis of strains isolated from different hosts. Elucidation of the genomic diversity and investigation of the functional differences in the Australian population could provide valuable information towards disease control. In this study, isolates of T. basicola were investigated for genomic (internal transcribed spacers region), proteomic and cotton virulence level variations. Internal transcribed spacers sequence analysis revealed that isolates are grouped based on host of origin irrespective of geographical origin. At the proteome level a degree of diversity was apparent and hierarchical clustering analysis of the data also demonstrated a close correlation between the proteome and the host of origin. LC-MS/MS analysis and identification using cross-species similarity searching and de novo sequencing of host-specific differentially expressed proteins and the virulence-correlated proteome allowed successful identification of 43 spots. The majority were found to be involved in metabolism. Spots that were correlated with host and virulence differences included a hypothetical protein with a Rossman-fold NAD(P)(+)-binding protein domain, glyceraldehyde-3-phosphate dehydrogenase, arginase and tetrahydroxynaphthalene reductase.
User Published Research Ruan L, Pleitner A, Gänzle MG, McMullen LM. Solute transport proteins and the outer membrane protein NmpC contribute to heat-resistance of Escherichia coli AW1.7. Appl Environ Microbiol. 2011 Mar 11.
This study aimed to elucidate determinants of heat resistance in E. coli by comparing the composition of membrane lipids as well as gene expression in the heat resistant E. coli AW1.7 and the heat sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late-exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids compared to E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression of these two strains. Expression of selected genes from different functional group was quantified by quantitative PCR (q-PCR). DnaK, 30S and 50S risobomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. SDS-PAGE analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50 - 1000 fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7 but heat resistance in this strain is dependent on additional factors, which likely include the composition of membrane lipids as well as solute transport proteins.
User Published Research Karlsen C, Espelid S, Willassen NP, Paulsen SM. Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome. Dis Aquat Org 2011 Feb. 93:215-223
Aliivibrio salmonicida is the aetiological agent of cold water vibriosis affecting farmed fish species, a disease that today is fully controlled by vaccination. However, the molecular mechanisms behind the successful vaccine are largely unknown. In order to gain insight into the possible mechanisms of A. salmonicida vaccines, we report here the profiles of both the outer membrane and secreted subproteomes of A. salmonicida LFI315. The 2 subproteomes were resolved by 2-dimensional electrophoresis that identified a total of 82 protein entries. Monoclonal antibodies specific to an unidentified protein antigen were utilized in the immunoproteomic analysis of both outer membrane proteins and extracellular proteins. The immunogenic protein was located in both subproteomes and identified as a 20 kDa peptidoglycan-associated lipoprotein (Pal). The identity of the antigen was verified by heterologous expression of the cloned A. salmonicida pal gene (VSAL_I1899). It is likely that the immunogenic Pal-like protein is among the constituents that act as a protective antigen in the successful vaccine used today. In view of this, it may be considered a potentially useful component in future vaccine development and pathogenicity studies.
User Published Research Bovi M, Carrizo ME, Capaldi S, Perduca M, Chiarelli LR, Galliano M, Monaco HL. Structure of a Lectin with Antitumoral Properties in King Bolete (Boletus edulis) Mushrooms. Glycobiology. 2011 Feb 8.
A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino, or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (Boletus edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142 amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.
User Published Research Ons S, Sterkel M, Diambra L, Urlaub H, Rivera-Pomar R. Neuropeptide precursor gene discovery in the Chagas disease vector Rhodnius prolixus. Insect Mol Biol. 2011 Feb;20(1):29-44.
We show a straightforward workflow combining homology search in Rhodnius prolixus genome sequence with cloning by rapid amplification of cDNA ends and mass spectrometry. We have identified 32 genes and their transcripts that encode a number of neuropeptide precursors leading to 194 putative peptides. We validated by mass spectrometry 82 of those predicted neuropeptides in the brain of R. prolixus to achieve the first comprehensive genomic, transcriptomic and neuropeptidomic analysis of an insect disease vector. Comparisons of available insect neuropeptide sequences revealed that the R. prolixus genome contains most of the conserved neuropeptides in insects, many of them displaying specific features at the sequence level. Some gene families reported here are identified for the first time in the order Hemiptera, a highly biodiverse group of insects that includes many human, animal and plant disease agents.
User Published Research Heinz A, Taddese S, Sippl W, Neubert RH, Schmelzer CE. Insights into the degradation of human elastin by matrilysin-1. Biochimie. 2011 Feb;93(2):187-94.
Human matrilysin-1 (MMP-7) is one of the most potent elastases besides macrophage elastase in the family of matrix metalloproteinases (MMPs). It has been reported to provide macrophages with the highest elastinolytic capacity and plays key roles in diseases such as emphysema and cancer. Describing the enzymatic turnover of matrix components helps to understand the molecular basis of disease processes. Hence, in this work, the cleavage behavior of MMP-7 with respect to its natural substrate human elastin was investigated using mass spectrometric (MS) techniques and molecular modeling. Elastin peptides in the range of 500-8000 Da released through the action of MMP-7 were analyzed and domains susceptible to proteolytic attack by MMP-7 were identified. MMP-7 was found to mainly cleave in N- and C-terminal regions of elastin's precursor, which involves linkages in domains encoded by exons 2, 3, 5-7, 26, and 30-33. In contrast, only few cleavages were found in the central part of the precursor and no cleavages in regions in elastin that are involved in cross-linking. MMP-7 shows a strong preference for Leu in P(1)' and also accepts Val, Gly, and Pro at this position, whereas Ala is not preferred at P(1)'. Analysis by molecular modeling revealed that not only the size of the amino acid residue in P(1)' but also the orientation of the neighboring P(1) residue and, thus, the orientation of the peptide bond that is cleaved influences the cleavage preference of MMP-7. Overall, this study provides an important insight into the degradation of human elastin by MMP-7 and may aid in the development of approaches to treat elastin-degrading diseases.
User Published Research Kakizaki I, Tatara Y, Majima M, Kato Y, Endo M. Identification of proteoglycan from salmon nasal cartilage. Arch Biochem Biophys. 2011 Feb 1;506(1):58-65.
Eurofibromatosis 2 or bilateral acoustic neurofibromatosis (NF2) is a severe autosomal dominant disorder characterized by the development of multiple tumors of the nervous system, including meningiomas, gliomas, neurofibromas, ependymomas, and particularly acoustic neuromas. Polymorphic DNA markers have revealed frequent loss of one copy of chromosome 22 in the tumor types associated with NF2. Family studies have demonstrated that the primary defect in NF2 is linked to DNA markers on chromosome 22, suggesting that it involves inactivation of a tumor suppressor gene. We have employed a combination of multipoint linkage analysis and examination of deletions in primary tumor specimens to precisely map the NF2 locus between flanking polymorphic DNA markers on chromosome 22. The 13-cM region bracketed by these markers corresponds to 13% of the genetic length of the long arm of chromosome 22 and is expected to contain less than 5 x 10(6) bp of DNA. The delineation of flanking markers for NF2 should permit accurate presymptomatic and prenatal diagnosis for the disorder and greatly facilitate efforts to isolate the defective gene on the basis of its location.
User Published Research Kaur P, Jost R, Sivasithamparam K, Barbetti MJ. Proteome analysis of the Albugo candida-Brassica juncea pathosystem reveals that the timing of the expression of defence-related genes is a crucial determinant of pathogenesis. J Exp Bot. 2011 Jan;62(3):1285-98.
White rust, caused by Albugo candida, is a serious pathogen of Brassica juncea (Indian mustard) and poses a potential hazard to the presently developing canola-quality B. juncea industry worldwide. A comparative proteomic study was undertaken to explore the molecular mechanisms that underlie the defence responses of Brassica juncea to white rust disease caused by the biotrophic oomycete Albugo candida. Nineteen proteins showed reproducible differences in abundance between a susceptible (RH 819) and a resistant variety (CBJ 001) of B. juncea following inoculation with A. candida. The identities of all 19 proteins were successfully established through Q-TOF MS/MS. Five of these proteins were only detected in the resistant variety and showed significant differences in their abundance at various times following pathogen inoculation in comparison to mock-inoculated plants. Among these was a thaumatin-like protein (PR-5), a protein not previously associated with the resistance of B. juncea towards A. candida. One protein, peptidyl-prolyl cis/trans isomerase (PPIase) isoform CYP20-3, was only detected in the susceptible variety and increased in abundance in response to the pathogen. PPIases have recently been discovered to play an important role in pathogenesis by suppressing the host cell's immune response. For a subset of seven proteins examined in more detail, an increase in transcript abundance always preceded their induction at the proteome level. These findings are discussed within the context of the A. candida-Brassica juncea pathosystem, especially in relation to host resistance to this pathogen
User Published Research Dekker LJ, Zeneyedpour L, Brouwer E, van Duijn MM, Sillevis Smitt PA, Luider TM. An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions. Anal Bioanal Chem. 2011 Jan;399(3):1081-91.
Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples.
User Published Research Matysiak J, Schmelzer CE, Neubert RH, Kokot ZJ. Characterization of honeybee venom by MALDI-TOF and nanoESI-QqTOF mass spectrometry. J Pharm Biomed Anal. 2011 Jan 25;54(2):273-8.
The aim of the study was to comprehensively characterize different honeybee venom samples applying two complementary mass spectrometry methods. 41 honeybee venom samples of different bee strains, country of origin (Poland, Georgia, and Estonia), year and season of the venom collection were analyzed using MALDI-TOF and nanoESI-QqTOF-MS. It was possible to obtain semi-quantitative data for 12 different components in selected honeybee venom samples using MALDI-TOF method without further sophisticated and time consuming sample pretreatment. Statistical analysis (ANOVA) has shown that there are qualitative and quantitative differences in the composition between honeybee venom samples collected over different years. It has also been demonstrated that MALDI-TOF spectra can be used as a "protein fingerprint" of honeybee venom in order to confirm the identity of the product. NanoESI-QqTOF-MS was applied especially for identification purposes. Using this technique 16 peptide sequences were identified, including melittin (12 different breakdown products and precursors), apamine, mast cell degranulating peptide and secapin. Moreover, the significant achievement of this study is the fact that the new peptide (HTGAVLAGV+Amidated (C-term), M(r)=822.53Da) has been discovered in bee venom for the first time.
User Published Research Lamy E, Graça G, da Costa G, Franco C, E Silva FC, Baptista ES, Coelho AV. Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet. Proteome Sci. 2010 Dec 15;8:65.
Background: Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry.
Results: Fifty-seven protein spots were excised from control group gels, and 21 different proteins were identified. With tannin consumption, the expression levels of one α-amylase isoform and one unidentified protein increased, whereas acidic mammalian chitinase and Muc10 decreased. Additionally, two basic spots that stained pink with Coomassie Brilliant Blue R-250 were newly observed, suggesting that some induced PRPs may remain uncomplexed or form soluble complexes with tannins.
Conclusion: This proteomic analysis provides evidence that other salivary proteins, in addition to tannin-precipitating proteins, are affected by tannin ingestion. Changes in the expression levels of the acidic mammalian chitinase precursor and in one of the 14 salivary α-amylase isoforms underscores the need to further investigate their role in tannin ingestion.
User Published Research Jing J, Sweedler JV, Cropper EC, Alexeeva V, Park JH, Romanova EV, Xie F, Dembrow NC, Ludwar BC, Weiss KR, Vilim FS. Feedforward compensation mediated by the central and peripheral actions of a single neuropeptide discovered using representational difference analysis. J Neurosci. 2010 Dec 8;30(49):16545-58.
Compensatory mechanisms are often used to achieve stability by reducing variance, which can be accomplished via negative feedback during homeostatic regulation. In principle, compensation can also be implemented through feedforward mechanisms where a regulator acts to offset the anticipated output variation; however, few such neural mechanisms have been demonstrated. We provide evidence that an Aplysia neuropeptide, identified using an enhanced representational difference analysis procedure, implements feedforward compensation within the feeding network. We named the novel peptide "allatotropin-related peptide" (ATRP) because of its similarity to insect allatotropin. Mass spectrometry confirmed the peptide's identity, and in situ hybridization and immunostaining mapped its distribution in the Aplysia CNS. ATRP is present in the higher-order cerebral-buccal interneuron (CBI) CBI-4, but not in CBI-2. Previous work showed that CBI-4-elicited motor programs have a shorter protraction duration than those elicited by CBI-2. Here we show that ATRP shortens protraction duration of CBI-2-elicited ingestive programs, suggesting a contribution of ATRP to the parametric differences between CBI-4-evoked and CBI-2-evoked programs. Importantly, because Aplysia muscle contractions are a graded function of motoneuronal activity, one consequence of the shortening of protraction is that it can weaken protraction movements. However, this potential weakening is offset by feedforward compensatory actions exerted by ATRP. Centrally, ATRP increases the activity of protraction motoneurons. Moreover, ATRP is present in peripheral varicosities of protraction motoneurons and enhances peripheral motoneuron-elicited protraction muscle contractions. Therefore, feedforward compensatory mechanisms mediated by ATRP make it possible to generate a faster movement with an amplitude that is not greatly reduced, thereby producing stability.
User Published Research Ibarz A, Costa R, Harrison AP, Power DM. Dietary keto-acid feed-back on pituitary activity in gilthead sea bream: effects of oral doses of AKG. A proteomic approach. Gen Comp Endocrinol. 2010 Dec 1;169(3):284-92.
The influence of a daily oral dose of alpha-ketoglutarate (AKG, 0.1 g/kg body weight), an intermediate metabolite in the Krebs cycle and a dietary additive, on the pituitary proteome of gilthead sea bream was determined by two-dimensional electrophoresis (2-DE). A high-resolution map of the sea bream pituitary proteome was generated. Proteins with a modified expression between Controls and AKG treated fish were further analysed by MALDI-TOF/TOF-MS and liquid chromatography combined with a nanoelectrospray (LC-MS/MS). The main changes in the proteome induced by AKG treatment were grouped. Metabolic proteins up-regulated with AKG supplementation included fructose-bis-phosphate aldolase, glyceraldehyde-phosphate dehydrogenase and malate dehydrogenase, all related to glucose metabolism (p<0.000). Protein folding related up-regulation with AKG supplementation included two isoforms of heat shock proteins as well as cyclophylin and chaperonin (p<0.000). An unexpected form of apolipoprotein-A-1 with lower molecular weight (15-16 kDa) was evidenced as being highly abundant in the pituitary proteome of Controls, yet it was down-regulated by AKG treatment. Finally, proteins found to be associated with regeneration of neural function namely cofilin and Vat-protein were up-regulated after AKG supplementation. The only hormone to be modified by AKG treatment was somatolactin, which was significantly down-regulated cf. Controls. In summary, these results provide evidence of a potential endocrine/metabolic regulatory loop activated by AKG supplementation
User Published Research Trabalon M, Carapito C, Voinot F, Martrette JM, Van Dorsselaer A, Gilbert C, Bertile F. Differences in Brachypelma albopilosa (Theraphosidae) hemolymph proteome between subadult and adult females. J Exp Zool A Ecol Genet Physiol. 2010 Dec 1;313(10):651-9.
The changes in the hemolymph proteome of mygalomorph Brachypelma albopilosa females were examined for the first time in relation to their developmental stage (subadult and adult period). Seven distinct subunits of hemocyanin (a, b, c, d, e, f, and g chains), as well as actin were clearly identified and their sequence partly characterized using a combination of one- and two-dimensional gel electrophoresis and mass spectrometry. The different structures determined along with possible post-translational modifications may reflect a role of hemocyanin in molting, immunity, and reproduction. In addition, despite no precise identification, additional peptide sequences from eight protein bands (four bands >200 kDa and four bands in the 95-200 kDa mass range) were determined. As reported in other spider species, the putative corresponding structures are the coagulogen protein and/or lipoproteins (HDL-1, HDL-2, VHDL) for which quantitative differences between adult and subadult individuals could be related to the molting process and/or cuticle lipid and protein composition according to the developmental stage.
User Published Research Antunes AM, Godinho AL, Martins IL, Oliveira MC, Gomes RA, Coelho AV, Beland FA, Marques MM. Protein adducts as prospective biomarkers of nevirapine toxicity. Chem Res Toxicol. 2010 Nov 15;23(11):1714-25.
Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against human immunodeficiency virus type-1 (HIV-1), mostly to prevent mother-to-child HIV-1 transmission in developing countries. Despite its clinical efficacy, NVP administration is associated with a variety of toxic responses that include hepatotoxicity and skin rash. Although the reasons for the adverse effects of NVP administration are still unclear, increasing evidence supports the involvement of metabolic activation to reactive electrophiles. In particular, Phase II activation of the NVP metabolite 12-hydroxy-NVP is thought to mediate NVP binding to bionucleophiles, which may be at the onset of toxicity. In the present study, we investigated the nature and specific locations of the covalent adducts produced in human serum albumin and human hemoglobin by reaction in vitro with the synthetic model electrophile 12-mesyloxy-NVP, used as a surrogate for the Phase II metabolite 12-sulfoxy-NVP. Multiple sites of modification were identified by two different mass spectrometry-based methodologies, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-TOF-TOF-MS). These two distinct methodologies, which in some instances afforded complementary information, allowed the identification of multiple adducts involving cysteine, lysine, tryptophan, histidine, serine, and the N-terminal valine of hemoglobin. Tryptophan, which is not a common site of covalent protein modification, was the NVP-modified amino acid residue detected in the two proteins and consistently identified by both LC-ESI-MS/MS and MALDI-TOF-TOF-MS. The propensity of tryptophan to react with the NVP-derived electrophile is further emphasized by the fact that human serum albumin possesses a single tryptophan residue, which suggests a remarkable selectivity that may be useful for biomonitoring purposes. Likewise, the NVP adduct with the terminal valine of hemoglobin, detected by LC-ESI-MS/MS after N-alkyl Edman degradation, appears as an easily assessed marker of NVP binding to proteins. Our results demonstrate the merits and complementarity of the two MS-based methodologies for the characterization of protein binding by NVP and suggest a series of plausible biomarkers of NVP toxicity that should be useful in the monitoring of toxicity effects in patients administered NVP.
User Published Research Sofiadis A, Dinets A, Orre LM, Branca RM, Juhlin CC, Foukakis T, Wallin G, Höög A, Hulchiy M, Zedenius J, Larsson C, Lehtiö J. Proteomic study of thyroid tumors reveals frequent up-regulation of the Ca2+ -binding protein S100A6 in papillary thyroid carcinoma. Thyroid. 2010 Oct;20(10):1067-76.
Background: The accurate diagnosis of thyroid tumors is challenging. Proteomics has emerged as a promising approach for the discovery of molecular diagnostic markers as a potential complement to routine diagnostics.
Methods: Protein fractions from 29 frozen thyroid tumor tissue samples (10 papillary carcinomas, 9 follicular carcinomas, and 10 follicular adenomas) as well as from normal thyroid tissue were analyzed by surface enhanced laser desorption/ionization time-of-flight mass spectrometry followed by validation by Western blotting and immunohistochemistry.
Results: A Ca2+ binding protein belonging to the S100 family, S100A6, was differentially expressed between papillary and follicular thyroid tumors. Moreover, two posttranslationally modified forms of S100A6 were observed and verified by liquid chromatography-coupled tandem mass spectrometry. Validation by Western blotting displayed a significantly higher expression of S100A6 in papillary thyroid carcinoma (PTC) in comparison with the other tumor groups or normal tissue (p<0.05). Immunohistochemical analysis on 98 tumors showed that PTC cases had a significantly stronger cytosolic staining and a larger proportion of stained nuclei than follicular tumors. BRAF gene mutation was not significantly associated with S100A6 protein levels.
Conclusion: This study supports a role of S100A6 in thyroid tumorigenesis and as a potential aid in the discrimination between follicular thyroid tumors and PTC.
User Published Research Collins JJ 3rd, Hou X, Romanova EV, Lambrus BG, Miller CM, Saberi A, Sweedler JV, Newmark PA. Genome-wide analyses reveal a role for peptide hormones in planarian germline development. PLoS Biol. 2010 Oct 12;8(10):e1000509.
Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.
User Published Research Twine SM, Tessier L, Kelly JF. Glycoprotein characterization. Methods Mol Biol. 2010;600:111-31.
Increasing numbers of studies are reporting the modification of prokaryotic proteins with novel glycans. These proteins are often associated with virulence factors of medically important pathogens. Herein, we describe the steps required to characterize prokaryotic glycoproteins by mass spectrometry, using flagellin isolated from Clostridium botulinum strain Langeland as an example. Both "top-down" and "bottom-up" approaches will be described for characterizing the purified glycoprotein at the whole protein and peptide levels. The preliminary steps toward structural characterization of novel prokaryotic glycans by mass spectrometry and NMR are also described.
User Published Research Romanova EV, Lee JE, Kelleher NL, Sweedler JV, Gulley JM. Mass spectrometry screening reveals peptides modulated differentially in the medial prefrontal cortex of rats with disparate initial sensitivity to cocaine. AAPS J. 2010 Sep;12(3):443-54.
To better understand why certain individuals are more vulnerable to cocaine abuse and addiction, we identify peptide markers associated with individual variation in sensitivity to the behavioral effects of cocaine. Previous studies in rats show that low, compared to high, cocaine responders are more sensitive to cocaine-induced behavioral plasticity (sensitization), exhibit enhanced conditioning to cocaine's rewarding effects, and are more motivated to self administer cocaine. In the current study, we combine matrix-assisted laser desorption/ionization mass spectrometry with multivariate statistical methods to analyze tissue extracts from rat dorsal striatum, nucleus accumbens, and medial prefrontal cortex (mPFC) to examine trends in peptide changes that coincide with behavioral phenotype. Peptide profiles of these three regions from individual animals were characterized via mass spectrometry. Resulting mass peaks that were statistically different between these groups were identified using principal component analysis. The mass peaks were then identified in pooled samples via multistage liquid chromatography mass spectrometry. A total of 74 peptides from 28 proteins were sequenced from defined brain regions. Statistically significant changes in peak intensities for seven peptides were found in the mPFC of rats given a single injection of 10 mg/kg cocaine, with low cocaine responders showing approximately 2-fold increase in peak intensities for the acetylated N terminus peptides of stathmin and Hint 1, as well as truncated ATP synthase. These results suggest that distinct peptide profiles in the mPFC are associated with individuals that exhibit reduced sensitivity to the behavioral effects of cocaine.
User Published Research VanDuijn MM, Dekker LJ, Zeneyedpour L, Smitt PA, Luider TM. Immune responses are characterized by specific shared immunoglobulin peptides that can be detected by proteomic techniques. J Biol Chem. 2010 Sep 17;285(38):29247-53.
In the adaptive immune response, immunoglobulins develop that bind specifically to the antigens to which the organism was exposed. Immunoglobulins may bind to known or unknown antigens in a variety of diseases and have been used in the past to identify novel antigens for use as a biomarker. We propose that the immunoglobulins themselves could also be used as biomarkers in antibody-mediated disease. In this proteomic study, rats were immunized with one of two purified antigens, and immunoglobulins from pre- and postimmune sera were analyzed with nano-LC coupled mass spectrometry. It was found that the two treatment groups could be distinguished based on cluster analysis of the immunoglobulin peptides from the immune sera. In addition, we identified 684 specific peptides that were differentially present in one of the two treated groups. We could find an amino acid sequence for 44% of the features in the mass spectra by combining database-driven and de novo sequencing techniques. The latter were essential for sequence identification, as the more common database-driven approach suffers from a poor representation of immunoglobulins in the available databases. Our data show that the development of immunoglobulins during an immune response is not a fully random process, but that instead selection pressures exist that favor the best binding amino acid sequences, and that this selection is shared between different animals. This finding implies that immunoglobulin peptides could indeed be a powerful and easily accessible class of biomarkers.
User Published Research Villar M, Torina A, Nuñez Y, Zivkovic Z, Marina A, Alongi A, Scimeca S, La Barbera G, Caracappa S, Vázquez J, Fuente Jde L. Application of highly sensitive saturation labeling to the analysis of differential protein expression in infected ticks from limited samples. Proteome Sci. 2010 Aug 12;8:43.
Background: Ticks are vectors of pathogens that affect human and animal health worldwide. Proteomics and genomics studies of infected ticks are required to understand tick-pathogen interactions and identify potential vaccine antigens to control pathogen transmission. One of the limitations for proteomics research in ticks is the amount of protein that can be obtained from these organisms. In the work reported here, individual naturally-infected and uninfected Rhipicephalus spp. ticks were processed using a method that permits simultaneous extraction of DNA, RNA and proteins. This approach allowed using DNA to determine pathogen infection, protein for proteomics studies and RNA to characterize mRNA levels for some of the differentially expressed proteins. Differential protein expression in response to natural infection with different pathogens was characterized by two-dimensional (2-D) differential in gel electrophoresis (DIGE) saturation labeling in combination with mass spectrometry analysis. To our knowledge, this is the first report of the application of DIGE saturation labeling to study tick proteins.
Results: Questing and feeding Rhipicephalus spp. adult ticks were collected in 27 farms located in different Sicilian regions. From 300 collected ticks, only 16 were found to be infected: R. sanguineus with Rickettsia conorii and Ehrlichia canis; R. bursa with Theileria annulata; and R. turanicus with Anaplasma ovis. The proteomic analysis conducted from a limited amount of proteins allowed the identification of host, pathogen and tick proteins differentially expressed as a consequence of infection.
Conclusion: These results showed that DIGE saturation labeling is a powerful technology for proteomics studies in small number of ticks and provided new information about the effect of pathogen infection in ticks.
User Published Research Boldo JT, do Amaral KB, Junges A, Pinto PM, Staats CC, Vainstein MH, Schrank A. Evidence of alternative splicing of the chi2 chitinase gene from Metarhizium anisopliae. Gene. 2010 Aug 15;462(1-2):1-7.
Metarhizium anisopliae is a filamentous fungus used in the biological control of arthropods and produces several chitinases in order to break the host cuticle chitin fibers. Chitinase function during fungal cell development and/or infection processes is also an important aspect when analyzing the life cycle of entomopathogens. The expression profile analysis of the endochitinase chi2 gene acquired by RT-PCR experiments indicated the presence of two different transcripts, suggesting the occurrence of alternative splicing in the chi2 gene. The presence of two transcripts, characterized by the removal or retention of the second 72 bp intron, was further confirmed by DNA sequencing, Northern blot and qRT-PCR. Furthermore, we detected the synthesis of two different proteins from the transcripts by two-dimensional Western blot and mass spectrometry analyses. This is the first reported occurrence of alternative splicing in M. anisopliae.
User Published Research Alves M, Chaves I, Carrilho D, Veloso M, Ricardo CP. Detection of novel trypsin inhibitors in the cotyledons of Phaseolus vulgaris seeds. J Plant Physiol. 2010 Jul 1;167(10):848-54.
Protease inhibitors play important roles in plants in association with stress. Trypsin inhibitors (TIs) in particular are known to act as protective agents against insect and pathogen attacks. By using the two-dimensional electrophoresis (2-DE) reverse zymography technique, we identified, from the crude extract of bean seeds, nine novel polypeptides that showed trypsin inhibitor activity. One of these polypeptide inhibitors yielded no homology in the database, which can be an indication that we are found a new protein with unique TI properties.
User Published Research Marsolais F, Pajak A, Yin F, Taylor M, Gabriel M, Merino DM, Ma V, Kameka A, Vijayan P, Pham H, Huang S, Rivoal J, Bett K, Hernández-Sebastià C, Liu Q, Bertrand A, Chapman R. Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway. J Proteomics. 2010 Jun 16;73(8):1587-600.
A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed.
User Published Research Dowd WW, Renshaw GM, Cech JJ Jr, Kültz D. Compensatory proteome adjustments imply tissue-specific structural and metabolic reorganization following episodic hypoxia or anoxia in the epaulette shark (Hemiscyllium ocellatum). Physiol Genomics. 2010 Jun;42(1):93-114.
The epaulette shark (Hemiscyllium ocellatum) represents an ancestral vertebrate model of episodic hypoxia and anoxia tolerance at tropical temperatures. We used two-dimensional gel electrophoresis and mass spectrometry-based proteomics approaches, combined with a suite of physiological measures, to characterize this species' responses to 1) one episode of anoxia plus normoxic recovery, 2) one episode of severe hypoxia plus recovery, or 3) two episodes of severe hypoxia plus recovery. We examined these responses in the cerebellum and rectal gland, two tissues with high ATP requirements. Sharks maintained plasma ionic homeostasis following all treatments, and activities of Na(+)/K(+)-ATPase and caspase 3/7 in both tissues were unchanged. Oxygen lack and reoxygenation elicited subtle adjustments in the proteome. Hypoxia led to more extensive proteome responses than anoxia in both tissues. The cerebellum and rectal gland exhibited treatment-specific responses to oxygen limitation consistent with one or more of several strategies: 1) neurotransmitter and receptor downregulation in cerebellum to prevent excitotoxicity, 2) cytoskeletal/membrane reorganization, 3) metabolic reorganization and more efficient intracellular energy shuttling that are more consistent with sustained ATP turnover than with long-term metabolic depression, 4) detoxification of metabolic byproducts and oxidative stress in light of continued metabolic activity, particularly following hypoxia in rectal gland, and 5) activation of prosurvival signaling. We hypothesize that neuronal morphological changes facilitate prolonged protection from excitotoxicity via dendritic spine remodeling in cerebellum (i.e., synaptic structural plasticity). These results recapitulate several highly conserved themes in the anoxia and hypoxia tolerance, preconditioning, and oxidative stress literature in a single system. In addition, several of the identified pathways and proteins suggest potentially novel mechanisms for enhancing anoxia or hypoxia tolerance in vertebrates. Overall, our data show that episodic hypoxic or anoxic exposure and recovery in the epaulette shark amplifies a constitutive suite of compensatory mechanisms that further prepares them for subsequent insults.
User Published Research de Costa D, Broodman I, Vanduijn MM, Stingl C, Dekker LJ, Burgers PC, Hoogsteden HC, Sillevis Smitt PA, van Klaveren RJ, Luider TM.Sequencing and Quantifying IgG Fragments and Antigen-Binding Regions by Mass Spectrometry. J Proteome Res. 2010 Jun 4;9(6):2937-45.
In cancer and autoimmune diseases, immunoglobulins with a specific molecular signature that could potentially be used as diagnostic or prognostic markers are released into body fluids. An immunomics approach based on this phenomenon relies on the ability to identify the specific amino acid sequences of the complementarity-determining regions (CDR) of these immunoglobulins, which in turn depends on the level of accuracy, resolution, and sensitivity that can be achieved by advanced mass spectrometry. Reproducible isolation and sequencing of antibody fragments (e.g., Fab) by high-resolution mass spectrometry (MS) from seven healthy donors revealed 43 217 MS signals: 225 could be associated with CDR1 peptides, 513 with CDR2 peptides, and 19 with CDR3 peptides. Seventeen percent of the 43 217 MS signals did not overlap between the seven donors. The Fab isolation method used is reproducible and fast, with a high yield. It provides only one Fab sample fraction for subsequent characterization by high-resolution MS. In 17% and 4% of these seven healthy donors, qualitative (presence/absence) and quantitative (intensity) differences in Fab fragments could be demonstrated, respectively. From these results, we conclude that the identification of a CDR signature as biomarker for autoimmune diseases and cancer without prior knowledge of the antigen is feasible.
User Published Research Casado-Vela J, Muries B, Carvajal M, Iloro I, Elortza F, Martinez-Ballesta MC. Analysis of Root Plasma Membrane Aquaporins from Brassica oleracea: Post-Translational Modifications, de novo Sequencing and Detection of Isoforms by High Resolution Mass Spectrometry. J Proteome Res. 2010 May 28.
Plasma membrane Intrinsic Proteins (PIPs), a subfamily of aquaporins, are ubiquitous membrane channel proteins that play a crucial role in water uptake in plants. The use of high-performance liquid chromatography coupled to tandem mass spectrometry (HPLCMS/MS) analysis of peptides has previously shown to be a valuable tool to differentiate among PIP homologs sharing a high sequence homology and also to characterize their post-translational modifications (PTMs). The recent introduction of mass spectrometers able to measure peptide mass with high mass accuracy, together with new alternative ways of peptide fragmentation allows the identification and characterization of proteins from non-sequenced organisms, such as broccoli. In this study we combined three endoproteases (trypsin, Glu-C and Lys-C) with HPLC-MS/MS analysis and two types of peptide fragmentations, CID (collision induced dissociation) and HCD (higher energy C-trap dissociation), to identify PIP isoforms and PTMs from broccoli roots. After de novo sequencing analysis, eight peptides showing homology to Arabidopsis thaliana PIPs were identified. Although Arabidopsis nomenclature of PIP isoforms has not been defined for broccoli, our results agree with the occurrence of seven AtPIP isoforms (PIP 1;1, PIP 1;2, PIP 1;3 and PIP2;2, PIP 2;3, PIP2;1 and PIP2;7) in broccoli roots, as compared to the plant model A. thaliana. To our knowledge, these results 20 represent the deepest characterization of the PIPs isolated from the roots of broccoli, a crop with increasing agronomical interest.
User Published Research Jung S, Fladerer C, Braendle F, Madlung J, Spring O, Nordheim A. Identification of a Novel Plasmopara Halstedii Elicitor Protein Combining De Novo Peptide Sequencing Algorithms and RACE-PCR. Proteome Sci. 2010 May 10;8:24.
Background: Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i).
Results: The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set.All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase.
Conclusion: Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.
User Published Research Takahashi N, Fujiu Y. Effects of the aminophenol analogue p-Dodecylaminophenol on mouse skin. J Invest Dermatol. 2010 May;130(5):1258-67.
p-Dodecylaminophenol (p-DDAP) was designed on the basis of structure-activity relationship studies on N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR), a synthetic derivative of retinoic acid (RA). p-DDAP exhibits antioxidative activities greater than those of RA and 4-HPR. RA shows biological effects in epidermal cells that include the inhibition of differentiation to the squamous phenotype. In the current study, we examined the effects of topical p-DDAP treatment on the skin of hairless mice as compared with those of RA treatment. p-DDAP caused an increase in epidermal thickness and decreased matrix metalloprotease and hyaluronidase activities in mouse skin tissues to the same extent that RA did. p-DDAP did not induce desquamation, erythema, or inflammatory cytokine expression as observed with RA treatment. Two-dimensional polyacrylamide gel electrophoresis patterns of proteins from skin treated with p-DDAP were distinct from those treated with RA. A protein induced by both p-DDAP and RA was identified as cytokeratin 16. p-DDAP did not elevate transcriptional activities of RA nuclear receptors. These results suggest that p-DDAP improves skin as potently as RA without causing the desquamation and erythema that the latter does. An increase in cytokeratin 16 expression might be essential for the effects of both p-DDAP and RA in skin healing and maintenance.
User Published Research Danelishvili L, Yamazaki Y, Selker J, Bermudez LE. Secreted Mycobacterium tuberculosis Rv3654c and Rv3655c proteins participate in the suppression of macrophage apoptosis. PLoS One. 2010 May 4;5(5):e10474.
Background: Inhibition of macrophage apoptosis by Mycobacterium tuberculosis has been proposed as one of the virulence mechanisms whereby the pathogen avoids the host defense. The mechanisms by which M. tuberculosis H37Rv strain suppress apoptosis and escapes human macrophage killing was investigated.
Methodology: The screening of a transposon mutant bank identified several mutants, which, in contrast to the wild-type bacterium, had impaired ability to inhibit apoptosis of macrophages. Among the identified genes, Rv3659c (31G12 mutant) belongs to an operon reminiscent of type IV pili. The Rv3654c and Rv3655c putative proteins in a seven-gene operon are secreted into the macrophage cytoplasm and suppress apoptosis by blocking the extrinsic pathway. The operon is highly expressed when the bacterium is within macrophages, compared to the expression level in the extracellular environment. Rv3654c recognizes the polypyrimidine tract binding Protein-associated Splicing Factor (PSF) and cleaves it, diminishing the availability of caspase-8. While M. tuberculosis inhibits apoptosis by the extrinsic pathway, the pathogen does not appear to affect the intrinsic pathway. Inactivation of the intrinsic pathway by pharmacologic agents afftects M. tuberculosis and induces cell necrosis. Likewise, inactivation of PSF by siRNA significantly decreased the level of caspase-8 in macrophages.
Conclusion: While M. tuberculosis inhibits the extrinsic pathway of apoptosis, it appears to activate the intrinsic pathway leading to macrophage necrosis as a potential exit strategy.
User Published Research Takahashi N, Fujiu Y. Cytokeratins 16 and 10 bind to retinoic acid covalently in skin tissue of mice. Br J Dermatol. 2010 May;162(5):974-9.
Background: Retinoic acid (RA) has various biological effects in mammalian cells and tissues. In epidermal cells, RA is an inhibitor of differentiation to the squamous phenotype. The molecular mechanisms underlying the effects of RA on epidermal cells and other cell types are mediated by RA nuclear receptors and retinoylation (acylation by RA) of proteins.
Objectives: To understand the components responsible for RA effects via RA nuclear receptors and retinoylation.
Methods: We examined for the first time RA-binding proteins in mouse skin in vivo by immunoblotting using anti-RA monoclonal antibodies and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Results: We identified eight RA-binding proteins in the skin of hairless mice that were increased by topical RA treatment. Three of these proteins were identified as cytokeratin 10, cytokeratin 16 and serum albumin.
Conclusion: These results raise the possibility that RA binding to cytokeratins in vivo may be involved in the effect of RA on keratinocytes in mouse skin."
User Published Research Silva JL, Barbosa JF, Bravo JP, Souza EM, Huergo LF, Pedrosa FO, Esteves E, Daffre S, Fernandez MA. Induction of a gloverin-like antimicrobial polypeptide in the sugarcane borer Diatraea saccharalis challenged by septic injury. Braz J Med Biol Res. 2010 May;43(5):431-6.
Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae) is an important pest for Brazilian sugarcane. In the present study, we detected two distinct spots in hemolymph from septic injured larvae (HDs1 and HDs2), which are separated by 2DE gel electrophoresis. Both spots were subjected to in-gel tryptic digestion and MALDI-TOF/TOF analysis, which revealed the sequence VFGTLGSDDSGLFGK present in both HDs1 and HDs2. This sequence had homology and 80% identity with specific Lepidoptera antimicrobial peptides called gloverins. Analyses using the ImageMaster 2D software showed pI 8.94 of the HDs1 spot, which is similar to that described to Hyalophora gloveri gloverin (pI 8.5). Moreover, the 14-kDa molecular mass of the spot HDs1 is compatible to that of gloverins isolated from the hemolymph of Trichoplusia ni, Helicoverpa armigera and H. gloveri. Antimicrobial assays with partially purified fractions containing the HDs1 and HDs2 polypeptides demonstrated activity against Escherichia coli. This is the first report of antimicrobial polypeptides in D. saccharalis, and the identification of these peptides may help in the generation of new strategies to control this pest.
User Published Research Miskevicha F, Davisb A, Leeprapaiwongb P, Gigantia V, Kostićb NM, Angelb LA. Metal Complexes as Artificial Proteases in Proteomics: A Palladium(II) Complex Cleaves Various Proteins in Solutions Containing Detergents. Journal of Inorganic Biochemistry, 2010
Most popular agents for site-specific protein cleavage are proteolytic enzymes. Because they become denatured and inactivated by detergents, enzymes are inconvenient for proteomic analysis of hydrophobic proteins which require detergents as solubilizing agents. We used cis-[Pd(en)(H2O)2]2+ (in which en represents ethylenediamine) as an artificial protease to effect cleavage of three bovine proteins, namely ubiquitin, β-casein, and serum albumin, in separate experiments. Cleavage took place in aqueous solutions containing 1.0% wt/vol of either 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Zwittergent 3–14 at 2.5 < pH < 2.9 and 55–60 °C for 3–72 h. Digests were separated by HPLC and analyzed by tandem mass spectrometry. Peptides were identified by de novo sequencing and matched against the bovine genome. Because cleavage by Pd(II) complexes is rather selective and therefore infrequent, 72% of the identified peptides in the digests contained more than 10 amino acid. Palladium(II) complexes hold promise as cleavage agents in proteomics studies of membrane proteins.
User Poster Research Chang MH, Wang JM, Chen SH. Identification of ZBRK1 homologue in mouse by a proteomic approach. Taiwan Proteomics Society 2010.
A novel human gene, ZBRK1, encodes a 58 kDa protein with an N-terminal KRAB(Kruppel-associated box) domain and eight central zinc fingers. The ZBRK1, also called as ZNF350, is known to interact with BRCA1 and induce its downstream genes. The evolutional gene addition and deletion make it hard to identify homologue protein between species. Until now, only bovine zinc finger protein 350 has been identified as human ZBRK1 homologue protein with their high similarity of their full sequences. In mouse, we failed to identify a homologue protein using previous strategy of highly similar amino acids sequences. Therefore, we develop a different strategy combined with ultra performance liquid chromatography (UPLC), mass spectrometry and bioinformatics in order to successfully overcome the genetic evolutional challenges and identify a homologue protein in mouse.
User Published Research Coumans JV, Moens PD, Poljak A, Al-Jaaidi S, Pereg L, Raftery MJ. Plant extract induced changes in the proteome of the soilborne pathogenic fungus Thielaviopsis basicola. Proteomics. 2010 Apr;10(8):1573-91.
Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. Analysis by LC-MS/MS of unique and differentially expressed spots and identification using, cross-species similarity searching and de novo sequencing allowed successful identification of 41 spots.
User Published Research Xie F, London SE, Southey BR, Annangudi SP, Amare A, Rodriguez-Zas SL, Clayton DF, Sweedler JV. The zebra finch neuropeptidome: prediction, detection and expression. BMC Biol. 2010 Apr 1;8:28.
Background: Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions.
Results: Complementary bioinformatic resources were integrated to survey the zebra finch genome, identifying 70 putative prohormones. Ninety peptides derived from 24 predicted prohormones were characterized using several MS platforms; tandem MS confirmed a majority of the sequences. Most of the peptides described here were not known in the zebra finch or other avian species, although homologous prohormones exist in the chicken genome. Among the zebra finch peptides discovered were several unique vasoactive intestinal and adenylate cyclase activating polypeptide 1 peptides created by cleavage at sites previously unreported in mammalian prohormones. MS-based profiling of brain areas required for singing detected 13 peptides within one brain nucleus, HVC; in situ hybridization detected 13 of the 15 prohormone genes examined within at least one major song control nucleus. Expression mapping also identified prohormone messenger RNAs in areas associated with spatial learning and social behaviours. Based on the whole-genome analysis, 40 prohormone probes were found on a commonly used zebra finch brain microarray. Analysis of these newly annotated transcripts revealed that six prohormone probes showed altered expression after birds heard song playbacks in a paradigm of song recognition learning; we partially verify this result experimentally.
Conclusion: The zebra finch peptidome and prohormone complement is now characterized. Based on previous microarray results on zebra finch vocal learning and synaptic plasticity, a number of these prohormones show significant changes during learning. Interestingly, most mammalian prohormones have counterparts in the zebra finch, demonstrating that this songbird uses similar biochemical pathways for neurotransmission and hormonal regulation. These findings enhance investigation into neuropeptide-mediated mechanisms of brain function, learning and behaviour in this model.
User Published Research Heinz A, Jung MC, Duca L, Sippl W, Taddese S, Ihling C, Rusciani A, Jahreis G, Weiss AS, Neubert RH, Schmelzer CE. Degradation of tropoelastin by matrix metalloproteinases--cleavage site specificities and release of matrikines. FEBS J. 2010 Apr;277(8):1939-56.
To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P(1)'. MMP-7 shows a strong preference for Leu at P(1)', which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P(1)'. All three MMPs showed a clear preference for Pro at P(3) that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing
User Published Research Jha SS, Danelishvili L, Wagner D, Maser J, Li YJ, Moric I, Vogt S, Yamazaki Y, Lai B, Bermudez LE. Virulence-related Mycobacterium avium subsp hominissuis MAV_2928 gene is associated with vacuole remodeling in macrophages. BMC Microbiol. 2010 Apr 1;10:100.
Background: Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.
Results: MAV_2928 gene is primarily upregulated upon phagocytosis. The transcriptional profile of macrophages infected with the wild-type bacterium and the mutant were examined using DNA microarray, which showed that the two bacteria interact uniquely with mononuclear phagocytes. Based on the results, it was hypothesized that the phagosome environment and vacuole membrane of the wild-type bacterium might differ from the mutant. Wild-type bacterium phagosomes expressed a number of proteins different from those infected with the mutant. Proteins on the phagosomes were confirmed by fluorescence microscopy and Western blot. The environment in the phagosome of macrophages infected with the mutant differed from the environment of vacuoles with M. hominissuis wild-type in the concentration of zinc, manganese, calcium and potassium.
Conclusion: The results suggest that the MAV_2928 gene/operon might participate in the establishment of bacterial intracellular environment in macrophages.
User Published Research Villar M, Ayllón N, Busby AT, Galindo RC, Blouin EF, Kocan E, Bonzón-Kulichenko E, Zivkovic Z, Almazán C, Torina A, Vázquez J, de la Fuente J. Expression of Heat Shock and Other Stress Response Proteins in Ticks and Cultured Tick Cells in Response to Anaplasma spp. Infection and Heat Shock. International Journal of ProteomicsVolume 2010 (2010), Article ID 657261, 11.
Ticks are ectoparasites of animals and humans that serve as vectors of Anaplasma and other pathogens that affect humans and animals worldwide. Ticks and the pathogens that they transmit have coevolved molecular interactions involving genetic traits of both the tick and the pathogen that mediate their development and survival. In this paper, the expression of heat shock proteins (HSPs) and other stress response proteins (SRPs) was characterized in ticks and cultured tick cells by proteomics and transcriptomics analyses in response to Anaplasma spp. infection and heat shock. The results of these studies demonstrated that the stress response was activated in ticks and cultured tick cells after Anaplasma spp. infection and heat shock. However, in the natural vector-pathogen relationship, HSPs and other SRPs were not strongly activated, which likely resulted from tick-pathogen coevolution. These results also demonstrated pathogen- and tick-specific differences in the expression of HSPs and other SRPs in ticks and cultured tick cells infected with Anaplasma spp. and suggested the existence of post-transcriptional mechanisms induced by Anaplasma spp. to control tick response to infection. These results illustrated the complexity of the stress response in ticks and suggested a function for the HSPs and other SRPs during Anaplasma spp. infection.
User Published Research Lopez-Llorca LV, Gómez-Vidal S, Monfort E, Larriba E, Casado-Vela J, Elortza F, Jansson HB, Salinas J, Martín-Nieto J. Expression of serine proteases in egg-parasitic nematophagous fungi during barley root colonization. Fungal Genet Biol. 2010 Apr;47(4):342-51.
Nematophagous fungi Pochonia chlamydosporia and P. rubescens colonize endophytically barley roots. During nematode infection, serine proteases are secreted. We have investigated whether such proteases are also produced during root colonization. Polyclonal antibodies against serine protease P32 of P. rubescens cross-reacted with a related protease (VCP1) of P. chlamydosporia, but not with barley proteases. These antibodies also detected an unknown ca. 65-kDa protein, labeled hyphae and appressoria of P. chlamydosporia and strongly reduced proteolytic activity of extracts from fungus-colonized roots. Mass spectrometry (MS) of 32-kDa protein bands detected peptides homologous to VCP1 only in Pochonia-colonized roots. Peptides homologous to barley serine carboxypeptidases were found in 65kDa bands of all roots. RT-PCR detected expression of VCP1 and a new P. chlamydosporia serine carboxypeptidase (SCP1) genes only in fungus-colonized roots. SCP1 shared limited sequence homology with VCP1 and P32. Expression in roots of proteases from nematophagous fungi could be greatly relevant for nematode biocontrol.
User Published Research Taddese S, Jung MC, Ihling C, Heinz A, Neubert RH, Schmelzer CE. MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III. Biochim Biophys Acta. 2010 Apr;1804(4):731-9.
Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly(775)-Leu(776)- in alpha-2 type I collagen and -Gly(775)-Ile(776)- in alpha-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III.
User Published Research Coumans JV, Moens PD, Poljak A, Al-Jaaidi S, Pereg L, Raftery MJ. Plant-extract-induced changes in the proteome of the soil-borne pathogenic fungus Thielaviopsis basicola. Proteomics. 2010 Apr;10(8):1573-91.
Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non-host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2-DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non-host status. Analysis by LC-MS/MS of unique and differentially expressed spots and identification using cross-species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several "morpho" proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B(6) is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.
User Published Research Muhammad Rizwan, Ingrid Miller, Fareeha Tasneem, Josef Böhm, Manfred Gemeiner and Ebrahim Razzazi-Fazeli Proteome analysis of Aspergillus ochraceus. Mycotoxin Research, 2010, Volume 26, Number 3, Pages 171-180
Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.
User Published Research Ibarz A, Martín-Pérez M, Blasco J, Bellido D, de Oliveira E, Fernández-Borràs J. Gilthead sea bream liver proteome altered at low temperatures by oxidative stress. Proteomics. 2010 Mar;10(5):963-75.
Gilthead sea bream exposed to the cold show multiple physiological alterations, particularly in liver. A typical cold-stress response was reproduced in gilthead sea bream acclimated to 20 degrees C (Warm group) when the water temperature was lowered to 8 degrees C (Cold group). After 10 days, thiobarbituric acid reactive substances in the liver had increased by 50%, and nitric oxide had increased twofold. This indicates that lipid peroxidation and oxidative stress had occurred. Protein profiles of liver from fish in warm and cold environments were obtained by 2-DE. Quantification of differential expression by matching spots showed that a total of 57 proteins were altered significantly. Many proteins were downregulated following cold exposure, including actin, the most abundant protein in the proteome; enzymes of amino acid metabolism; and enzymes with antioxidant capacity, such as betaine-homocysteine-methyl transferase, glutathione-S-transferase and catalase. Some proteins associated with protective action were upregulated at low temperatures, including peroxiredoxin, thioredoxin and lysozyme; as well as enzymes such as aldehyde dehydrogenase and adenosin-methionine synthetase. However, the upregulation of proteases, proteasome activator protein and trypsinogen-like protein indicated an increase in proteolysis. Increases in elongation factor-1alpha, the GAPDH oxidative form, tubulin and Raf-kinase inhibitor protein indicated oxidative stress and the induction of apoptosis. These data indicate that cold exposure induced oxidative damage in hepatocytes.
User Published Research Montes Nieto R, García-Barrera T, Gómez-Ariza JL, López-Barea J. Environmental monitoring of Domingo Rubio stream (Huelva Estuary, SW Spain) by combining conventional biomarkers and proteomic analysis in Carcinus maenas. Environ Pollut. 2010 Feb;158(2):401-8.
Element load, conventional biomarkers and altered protein expression profiles were studied in Carcinus maenas crabs, to assess contamination of "Domingo Rubio" stream, an aquatic ecosystem that receives pyritic metals, industrial contaminants, and pesticides. Lower antioxidative activities - glucose-6-phosphate and 6-phosphogluconate dehydrogenases, catalase - were found in parallel to higher levels of damaged biomolecules - malondialdehyde, oxidized glutathione -, due to oxidative lesions promoted by contaminants, as the increased levels of essential - Zn, Cu, Co - and nonessential - Cr, Ni, Cd - elements. Utility of Proteomics to assess environmental quality was confirmed, especially after considering the six proteins identified by de novo sequencing through capLC-muESI-ITMS/MS and homology search on databases. They include tripartite motif-containing protein 11 and ATF7 transcription factor (upregulated), plus CBR-NHR-218 nuclear hormone receptor, two components of the ABC transporters and aldehyde dehydrogenase (downregulated). These proteins could be used as novel potential biomarkers of the deleterious effects of pollutants present in the area.
User Published Research Tannu NS, Howell LL, Hemby SE. Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration. Mol Psychiatry. 2010 Feb;15(2):185-203.
The reinforcing effects and long-term consequences of cocaine self-administration have been associated with brain regions of the mesolimbic dopamine pathway, namely the nucleus accumbens (NAc). Studies of cocaine-induced biochemical adaptations in rodent models have advanced our knowledge; however, unbiased detailed assessments of intracellular alterations in the primate brain are scarce, yet essential, to develop a comprehensive understanding of cocaine addiction. To this end, two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare changes in cytosolic protein abundance in the NAc between rhesus monkeys self-administering cocaine and controls. Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser-desorption ionization time-of-flight time-of-flight (MALDI-TOF-TOF). In total, 1098 spots were subjected to statistical analysis with 22 spots found to be differentially abundant of which 18 proteins were positively identified by mass spectrometry. In addition, approximately 1000 protein spots were constitutively expressed of which 21 proteins were positively identified by mass spectrometry. Increased levels of proteins in the cocaine-exposed monkeys include glial fibrillary acidic protein, syntaxin-binding protein 3, protein kinase C isoform, adenylate kinase isoenzyme 5 and mitochondrial-related proteins, whereas decreased levels of proteins included beta-soluble N-ethylmaleimide-sensitive factor attachment protein and neural and non-neural enolase. Using a complimentary proteomics approach, the differential expression of phosphorylated proteins in the cytosolic fraction of these subjects was examined. Two-dimensional gel electrophoresis (2DGE) was followed by gel staining with Pro-Q Diamond phosphoprotein gel stain, enabling differentiation of approximately 150 phosphoprotein spots between the groups. Following excision and trypsin digestions, MALDI-TOF-TOF was used to confirm the identity of 15 cocaine-altered phosphoproteins. Significant increased levels were detected for gamma-aminobutyric acid type A receptor-associated protein 1, 14-3-3 gamma-protein, glutathione S-transferase and brain-type aldolase, whereas significant decreases were observed for beta-actin, Rab GDP-dissociation inhibitor, guanine deaminase, peroxiredoxin 2 isoform b and several mitochondrial proteins. Results from these studies indicate coordinated dysregulation of proteins related to cell structure, signaling, metabolism and mitochondrial function. These data extend and compliment previous studies of cocaine-induced biochemical alterations in human postmortem brain tissue, using an animal model that closely recapitulates the human condition and provide new insight into the molecular basis of the disease and potential targets for pharmacotherapeutic intervention.
User Published Research Boerjan B, Cardoen D, Bogaerts A, Landuyt B, Schoofs L, Verleyen P. Mass spectrometric profiling of (neuro)-peptides in the worker honeybee, Apis mellifera. Neuropharmacology. 2010 Jan;58(1):248-58.
The honeybee is the economically most important beneficial insect and a model for studying immunity, development and social behavior. Hence, this species was selected for genome sequencing and annotation. An intensive interplay between bioinformatics and mass spectrometry (MS) resulted in the annotation of 36 neuropeptide genes (Hummon et al., 2006). Exactly 100 peptides were demonstrated by a variety of MS techniques. In this follow-up study we dissected and analysed separately all ganglia of the central nervous system (CNS) of adult worker bees in three repeats. The combined MALDI-TOF spectra enabled the accurate mapping of 67 peptides, encoded by 20 precursors. We also demonstrated the expression of an additional but already predicted peptide. In addition to putative bioactive peptides we also list and discuss spacer peptides, propeptides and truncated peptides. The majority of such peptides have a more restricted distribution pattern. Their presence provides some information on the precursor turnover and/or the location of neural cell bodies in which they are produced. Of a given precursor, the (neuro)-peptides with the widest distribution pattern are likely to be the best candidates to interact with receptors. The separate analysis of a neuroendocrine complex and the mushroom body yields suggestions as to which (neuro)-peptides might act as hormones and which neuropeptides might be involved in the complex spectrum of non-hormone driven honeybee behaviour, at these sites. Our data complement immunohistochemical studies of (neuro)-peptides in the honeybee, and form a reference for comparative studies in other insect or arthropod models, in particular in the light of recent or upcoming genome projects. Finally, they also form a firm basis for physiological, functional and/or differential peptidomics studies in the honeybee.
User Published Research Blouin AG, Greenwood DR, Chavan RR, Pearson MN, Clover GR, MacDiarmid RM, Cohen D. A generic method to identify plant viruses by high-resolution tandem mass spectrometry of their coat proteins. J Virol Methods. 2010 Jan;163(1):49-56.
Although a number of protocols have been developed for detection of viruses at the genus or family level, universal approaches to detect and identify unknown viruses are still required. High-resolution tandem mass spectrometry was used to identify accurately peptide masses and their constituent sequences from partially purified plant virus preparations. Analysis of the peptide fragment masses against a virus database using pattern-matching algorithms identified sequences with homology to known virus peptides and also predicted peptides using de novo sequence analysis. This method provided sufficient information to confirm the identity of two known viruses that were included as controls (Cucumber mosaic virus and Tomato spotted wilt virus) and to identify unknown viruses in six viral isolates. The unknown viruses have been identified as four common viruses (Alfalfa mosaic virus, Tobacco streak virus, Citrus leaf blotch virus and Ribgrass mosaic virus), and two novel viruses (a potexvirus and a vitivirus). The identification of viruses from five distinct families by the tandem mass spectrometric determination of their coat protein demonstrates that this is a useful method for initial virus identification. This method, complemented with molecular or immunological procedures, provides a rapid and convenient way to identify both known and novel plant viruses.
User Published Research Lee SS, Lim J, Tan S, Cha J, Yeo SY, Agnew HD, Heath JR. Accurate Maldi-TOF-TOF Sequencing of One-Bead-One Compound peptide Libraries with Application to the identification of Multiligand protein Affiinity agents using in Siti click Chemistry Screening. Anal Chem. 2010 Jan 15;82(2):672-9.
Combinatorial one-bead-one-compound (OBOC) peptide libraries are widely used for affinity screening, and the sequencing of peptides from hit beads is a key step in the process. For rapid sequencing, CNBr cleavage of the peptides from the beads, followed by de novo sequencing by MALDI-TOF/TOF, is explored.
User Published Research Gay M, Carrascal M, Gorga M, Parés A, Abian J. Characterization of peptides and proteins in commercial HSA solutions. . Proteomics. 2010 Jan;10(2):172-81.
HSA solutions account for 14% of the world market for plasma products. Albumin is indicated for reestablishing and maintaining circulatory volume in situations resulting from traumatic shock, surgery, or blood loss. Albumin is also used in extracorporeal liver support devices that perform blood dialysis against this protein. However, the protein composition of therapeutic albumin is only partially known. We performed an exhaustive analysis of albumin composition using a proteomic approach. Low abundance proteins and peptides in these samples were concentrated using a strong anion exchange resin. The absorbed material was eluted with a stepwise gradient of ammonium trifluoroacetate and the protein fraction was digested and analyzed by multidimensional liquid chromatography coupled to ESI-MS/MS using a linear ion trap. A total of 1219 peptides corresponding to 141 proteins different from albumin were identified with a false discovery rate <1%. Near 50% of these proteins have been described previously as forming part of the albuminome. Some of these proteins are proteases (kallikrein) or protease inhibitors (kininogen and SRPK1) or have relevant functions in cell surface adhesion (selectin, cadherins, and ICAMs) or in immunity and defense (molecules of the complement system and attractin). Characterization of these proteins and peptides is crucial in order to understand the therapeutic and possible deleterious effects of albumin therapies, in which this solution is infused to treat different pathological conditions.
User Published Research Ferrari F, Fumagalli M, Profumo A, Viglio S, Sala A, Dolcini L, Temporini C, Nicolis S, Merli D, Corana F, Casado B, Iadarola P. Deciphering the proteomic profile of rice (Oryza sativa) bran: a pilot study. Electrophoresis. 2009 Dec;30(23):4083-94.
The exact knowledge of the qualitative and quantitative protein components of rice bran is an essential aspect to be considered for a better understanding of the functional properties of this resource. Aim of the present investigation was to extract the largest number of rice bran proteins and to obtain their qualitative characterization. For this purpose, three different extraction protocols have been applied either on full-fat or on defatted rice bran. Likewise, to identify the highest number of proteins, MS data collected from 1-DE, 2-DE and gel-free procedures have been combined. These approaches allowed to unambiguously identify 43 proteins that were classified as signalling/regulation proteins (30%), proteins with enzymatic activity (30%), storage proteins (30%), transfer (5%) and structural (5%) proteins. The fact that all extraction and identification procedures have been performed in triplicate with an excellent reproducibility provides a rationale for considering the platform of proteins shown in this study as the potential proteome profile of rice bran. It also represents a source of information to evaluate better the qualities of rice bran as food resource.
User Published Research Ozaki T, Yamashita T, Ishiguro S. Mitochondrial m-calpain plays a role in the release of truncated apoptosis-inducing factor from the mitochondria. Biochim Biophys Acta. 2009 Dec;1793(12):1848-59.
Calpains, calcium-dependent cysteine proteases, are involved in a variety of cellular processes. We have reported on the characteristics of mitochondrial mu-calpain and have shown that ERp57-associated mitochondrial mu-calpain cleaves the apoptosis-inducing factor (AIF) to a truncated form (tAIF). In addition, we found an unknown mitochondrial calpain. In this study, we identified and characterized this undescribed mitochondrial calpain in rat liver mitochondrial intermembrane space. The mitochondrial mu- and unknown calpains were separated by DEAE-Sepharose column chromatography. We immunoprecipitated the unknown calpain with anti-calpain small subunit and identified it as calpain 2 (m-calpain large subunit) by nanoflow-LC-MS/MS analysis and database searching. Because the identified mitochondrial calpain was stained with anti-m-calpain large subunit antibody, we named it mitochondrial m-calpain. The Ca(2+) dependency of mitochondrial m-calpain was similar to that of cytosolic m-calpain. Immunoprecipitation analyses showed that mitochondrial m-calpain is associated with a 75-kDa glucose-regulated protein, a member of the heat shock protein 70 family. We also investigated the involvement of mitochondrial m-calpain in the release of tAIF from mitochondria. Calpain inhibitor, PD150606, an anti-voltage-dependent anion channel (VDAC), and anti-Bax antibodies prevented the release of tAIF from mitochondria. In addition, we found that mitochondrial m-calpain truncated VDAC in Ca(2+)-dependent manner. This cleavage of VDAC promotes the mitochondrial accumulation of Bax and the release of tAIF from mitochondria. The accumulated Bax in mitochondrial outer membrane was co-immunoprecipitated with VDAC. Our results demonstrated that mitochondrial m-calpain plays a role in the release of tAIF from mitochondria by cleaving VDAC, and tAIF is released through VDAC-Bax pores.
User Published Research Dolcini L, Sala A, Campagnoli M, Labò S, Valli M, Visai L, Minchiotti L, Monaco HL, Galliano M. Identification of the amniotic fluid insulin-like growth factor binding protein-1 phosphorylation sites and propensity to proteolysis of the isoforms. FEBS J. 2009 Oct;276(20):6033-46.
Insulin-like growth factor binding protein-1 (IGFBP-1) is the major secreted protein of human decidual cells during gestation and, as a modulator of insulin-like growth factors or by independent mechanisms, regulates embryonic implantation and growth. The protein is phosphorylated and this post-translational modification is regulated in pregnancy and represents an important determinant of its biological activity. We have isolated, from human normal amniotic fluid collected in the weeks 16-18, the intact nonphosphorylated IGFBP-1 and five electrophoretically distinct phosphoisoforms and have determined their in vivo phosphorylation state. The unmodified protein was the most abundant component and mono-, bi-, tri- and tetraphosphorylated forms were present in decreasing amounts. The phosphorylation sites of IGFBP-1 were identified by liquid chromatography-tandem mass spectrometry analysis of the peptides generated with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. Five serines were found to be phosphorylated and, of these, four are localized in the central, weakly conserved, region, at positions 95, 98, 101 and 119, whereas one, Ser169, is in the C-terminal domain. The post-translational modification predominantly involves the hydrophilic stretch of amino acids representing a potential PEST sequence (proline, glutamic acid, serine, threonine) and our results show that the phosphorylation state influences the propensity of IGFBP-1 to proteolysis.
User Published Research Dahal D, Heintz D, Van Dorsselaer A, Braun HP, Wydra K. Pathogenesis and stress related, as well as metabolic proteins are regulated in tomato stems infected with Ralstonia solanacearum. Plant Physiol Biochem. 2009 Sep;47(9):838-46.
A comparative proteome analysis was initiated to systematically investigate the physiological response of tomato (Solanum lycopersicum) to infection with Ralstonia solanacearum, causal agent of bacterial wilt. Plants of the susceptible tomato recombinant inbred line NHG3 and the resistant NHG13 were either infected or not infected with R. solanacearum and subsequently used for proteome analysis. Two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) allowed the separation of about 650-690 protein spots per analysis. Twelve proteins were of differential abundance in susceptible plants in response to bacterial infection, while no differences were observed in the resistant genotype. LC-MS/MS analysis of these spots revealed 12 proteins, six of which were annotated as plant and six as bacterial proteins. Among the plant proteins, two represent pathogenesis related (PR) proteins, one stress response protein, one enzyme of carbohydrate and energy metabolism, and one hypothetical protein. A constitutive difference between resistant and susceptible lines was not found.
User Published Research Chora S, Starita-Geribaldi M, Guigonis JM, Samson M, Roméo M, Bebianno MJ. Effect of cadmium in the clam Ruditapes decussatus assessed by proteomic analysis. Aquat Toxicol. 2009 Oct 4;94(4):300-8.
Cadmium, an environmental stressor due to its toxicity, persistence and accumulation in biota, is widespread in the aquatic environment. Cadmium accumulation kinetics have revealed that Ruditapes decussatus has a high affinity to this metal. Proteomics is an effective tool to evaluate the toxic effects of contaminants. The aim of this study was to investigate the Cd effects in the gill and digestive gland of the sentinel species R. decussatus. Protein expression profiles (PEPs) in the clam tissues exposed to Cd (40 microg l(-1), 21 days) were compared to unexposed ones. Cd induces major changes in tissue-specific protein expression profiles in gill and digestive gland. This tissue dependent response results mainly from differences in Cd accumulation, protein inhibition and/or autophagy. An overall decrease of protein spots was detected in both treated tissues, being higher in gill. Some of the spots more drastically altered after pollutants exposure were excised and nine were identified by micro liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins identified by homology search in databases included: three proteins (8-fold) up-regulated, one down-regulated, four suppressed and one induced. Cd induces major changes in proteins involved in cytoskeletal structure maintenance (muscle-type actin, adductor muscle actin and beta-tubulin), cell maintenance (Rab GDP) and metabolism (ALDH and MCAD, both identified by de novo sequencing) suggesting potential energetic change. They provide a valuable knowledge of Cd effects at biochemical and molecular levels in the gill and digestive gland of R. decussatus.
User Published Research Weber D, Cléroux C, Godefroy SB. Emerging analytical methods to determine gluten markers in processed foods--method development in support of standard setting. Anal Bioanal Chem. 2009 Sep;395(1):111-7.
The availability of analytical methods to detect and determine levels of markers of priority allergens in foods is of the utmost importance to support standard setting initiatives, the development of compliance and enforcement activities, as well as to provide guidance to industry on implementation of quality control practices, ensuring the effectiveness of allergen-related sanitation techniques. This paper describes the development and implementation of a mass-spectrometry-based technique to determine markers for individual sources of gluten in beer products. This methodology was shown to answer the requirements of Health Canada's proposed labeling standard for individual gluten source declaration, in order to achieve its policy objectives (i.e., protection of sensitive consumers, while promoting choice). Minimal sample work-up was required and the results obtained by ELISA were further complemented using the LC-MS/MS method. This paper aims to demonstrate the feasibility of alternative techniques to ELISA-based methodologies to determine allergen and gluten markers in food.
User Published Research Alonso-del-Rivero M, Trejo SA, Rodríguez de la Vega M, González Y, Bronsoms S, Canals F, Delfín J, Diaz J, Aviles FX, Chávez MA. A novel metallocarboxypeptidase-like enzyme from the marine annelid Sabellastarte magnifica--a step into the invertebrate world of proteases. FEBS J. 2009 Sep;276(17):4875-90.
After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhibitor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N-terminal and internal sequence homologies with M14 metallocarboxypeptidase-like enzymes, as determined by MS and automated Edman degradation. The enzyme contains one atom of Zn per molecule, is activated by Ca2+ and is drastically inhibited by the metal chelator 1,10-phenanthroline, as well as by excess Zn2+ or Cu2+, but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallocarboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase-A synthetic substrates, such as those with hydrophobic residues at the C-terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O-like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metallocarboxypeptidases family (assignable to the M14A or pancreatic-like subfamily) with a wider specificity that has not been described previously.
User Published Research Casanovas A, Carrascal M, Abián J, López-Tejero MD, Llobera M. Discovery of lipoprotein lipase pI isoforms and contributions to their characterization. J Proteomics. 2009 Aug 20;72(6):1031-9.
Lipoprotein lipase (LPL) plays a pivotal role in lipid metabolism and is implicated in several pathophysiological conditions. A large number of LPL studies have been performed in rat, although the amount of information derived from direct study of the protein in this species is limited. Here we attempted to examine possible modifications of LPL using proteomic tools. By combining high-resolution two-dimensional gel electrophoresis and Western blot with biological mass spectrometry we demonstrate the coexistence of multiple LPL pI isoforms in rat heart. We studied the origin of this pI heterogeneity by: (1) comparison with the 2D pattern of LPL from post-heparin rat plasma (as a source of mature LPL); (2) protein dephosphorylation; (3) protein deglycosylation; and (4) partial sequencing of LPL isoforms. The results reveal that LPL pI heterogeneity does not correspond to different stages of intracellular maturation or protein phosphorylation. It can be partially explained by glycosylation, although other post-translational modifications must also be involved. We also report the first partial sequence to be obtained from direct study of rat LPL protein. These findings should be the basis for further research aimed at identifying the molecular differences between LPL isoforms and exploring their potential functional implications.
User Published Research Roman Y, Bed'hom B, Guillot A, Levrier J, Chaste-Duvernoy D, Bomsel-Demontoy MC, Jalme MS. Identification of apolipoprotein A-I in the alpha-globulin fraction of avian plasma. Vet Clin Pathol. 2009 Jun;38(2):206-12.
Background: Plasma protein electrophoresis is frequently used in birds as a tool for the diagnosis and monitoring of disease. Identification of proteins in individual peaks can help improve our understanding of changes in protein concentration in physiologic and pathologic conditions.
Objective: The aim of this study was to verify the presence and identity the protein(s) in the prominent alpha-globulin peak of orange-winged parrots (Amazona amazonica), black kites (Milvus migrans), and rock pigeons (Columba livia).
Methods: Heparinized plasma samples were obtained from 12 birds of each species. Agarose gel electrophoresis and total protein concentration were determined using standard techniques. One plasma sample from each species was then electrophoresed using high-resolution agarose gels to isolate the alpha-globulin band. Gel strips were digested in trypsin and peptides were extracted and analyzed using liquid chromatography with tandem mass spectrometry. De novo sequencing was used to identify the protein based on homology scoring against a protein database.
Results: Electrophoresis verified the presence of a single prominent alpha-globulin peak, usually in the alpha(1)-region, that had a median concentration of 9.4 g/L (range, 2.1-11.7 g/L, 21.6% of total protein) in parrots, 12.2 g/L (10.4-13.2 g/L, 35.9%) in kites, and 10.7 g/L (9.0-11.5 g/L, 40.0%) in pigeons. Mass spectrometry and sequencing analysis unequivocally identified the protein as a mature circulating form of apolipoprotein A-I (apo A-I) in all 3 species.
Conclusions: Apo A-I accounts for the prominent alpha-globulin peak and comprises a major proportion of total protein concentration in diverse avian species. As a high-density lipoprotein and negative acute phase protein with a pivotal role in cholesterol homeostasis, further study is warranted to determine the significance of changes in apo A-I concentration in avian electrophoretograms.
User Published Research Lindhout T, Lau PC, Brewer D, Lam JS. Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in Pseudomonas aeruginosa PAO1 via modulation of cell surface attachment. Microbiology. 2009 Oct;155(Pt 10):3449-60.
In many Gram-negative bacterial species, rough strains producing truncated lipopolysaccharide (LPS) generally exhibit defects in motility compared with smooth strains. However, the role that LPS plays in bacterial motility is not well understood. The goal of this study was to examine the relationship between LPS defects and motility of Pseudomonas aeruginosa. P. aeruginosa wild-type strain PAO1 and three isogenic mutants with defects in the rmlC, migA and wapR genes and producing truncated core oligosaccharide were investigated in terms of motility, attachment to glass and flagella expression. Compared with the wild-type, the three mutants showed significant retardation in both swarming motility on 0.5 % soft-agar plates and swimming motility on 0.3 % soft-agar plates. Moreover, attachment to abiotic surfaces was observed to be stronger in these mutants. The assembly of flagella appeared to be intact in these strains and the ability of individual cells to swim was unaffected. Flagellin proteins prepared from mutants rmlC and rmd, defective in the production of TDP-l-rhamnose and GDP-d-rhamnose, respectively, were compared and a change in molecular mass was observed only in the rmlC mutant. These data indicated that l-rhamnose, and not its enantiomer, d-rhamnose, is incorporated into the flagellin glycan of P. aeruginosa PAO1. The nucleotide-activated sugar precursor TDP-l-rhamnose is therefore shared between LPS biosynthesis and flagellin glycosylation in P. aeruginosa PAO1. Our results suggest that although biochemical precursors are shared by LPS and flagellin glycan biosynthesis, LPS truncations probably alter flagella-mediated motility in P. aeruginosa by modulating cell-surface attachment but not flagella synthesis.
User Published Research Oladiran A, Belosevic M. Trypanosoma Carassii Hsp70 Increases Expression of Inflammatory Cytokines and Chemokines in Macrophages of the Goldfish (Carassius auratus L.). Dev Comp Immunol. 2009 Oct;33(10):1128-36.
We report on the cloning and characterization of Trypanosoma carassii 70 KDa heat shock protein (hsp70). T. carassii hsp70 was secreted/excreted into culture medium in vitro and was recognized by sera from infected fish. Recombinant hsp70 (rhsp70) activated goldfish macrophages and stimulated the production of pro-inflammatory cytokines including interferon gamma (IFNgamma), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, (IL)-12 and chemokines CCL-1 and CXCL-8 (IL-8). T. carassii hsp70-induced cytokine expression was abrogated by pronase treatment of macrophages confirming the existence of receptor(s) on goldfish macrophage surface that recognize parasite molecule. Parasite hsp70 also up-regulated the expression inducible nitric oxide synthase (iNOS) isoforms A and B and induced a strong nitric oxide response of goldfish macrophages.
User Poster Research Dauly C, Escoubas P, Nicholson G, King G, Hornshaw M. Nanoscale characterization of spider venom peptides by high-resolution LC-MS/MS analysis. IMSC 2009.
Animal venoms are natural libraries of biologically active peptides. They encompass a wide variety of structures and pharmacological activities and represent an enormous resource of novel molecules to be used as insecticide, therapeutic and drug models. Venom profiling can be used for species identification and to indicate the presence of potentially unknown toxins. Here we demonstrate that de novo sequencing at the nanoscale level is applicable to venomics research.
User Published Research Portelius E, Price E, Brinkmalm G, Stiteler M, Olsson M, Persson R, Westman-Brinkmalm A, Zetterberg H, Simon AJ, Blennow K. A Novel Pathway for Amyloid Precursor Protein Processing. Neurobiol Aging. 2009 Jul 13.
Amyloid precursor protein (APP) can be proteolytically processed along two pathways, the amyloidogenic that leads to the formation of the 40-42 amino acid long Alzheimer-associated amyloid beta (Abeta) peptide and the non-amyloidogenic in which APP is cut in the middle of the Abeta domain thus precluding Abeta formation. Using immunoprecipitation and mass spectrometry we have shown that Abeta is present in cerebrospinal fluid (CSF) as several shorter isoforms in addition to Abeta1-40 and Abeta1-42. To address the question by which processing pathways these shorter isoforms arise, we have developed a cell model that accurately reflects the Abeta isoform pattern in CSF. Using this model, we determined changes in the Abeta isoform pattern induced by alpha-, beta-, and gamma-secretase inhibitor treatment. All isoforms longer than and including Abeta1-17 were gamma-secretase dependent whereas shorter isoforms were gamma-secretase independent. These shorter isoforms, including Abeta1-14 and Abeta1-15, were reduced by treatment with alpha- and beta-secretase inhibitors, which suggests the existence of a third and previously unknown APP processing pathway involving concerted cleavages of APP by alpha- and beta-secretase.
User Published Research Cheng Z, Wei YYC, Sung WWL, Glick BR, McConkey BJ. Proteomic analysis of the response of the plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress. Proteome Sci. 2009; 7: 18.
Background: Plant growth-promoting bacteria can alleviate the inhibitory effects of various heavy metals on plant growth, via decreasing levels of stress-induced ethylene. However, little has been done to detect any mechanisms specific for heavy metal resistance of this kind of bacteria. Here, we investigate the response of the wild-type plant growth-promoting bacterium Pseudomonas putida UW4 to nickel stress using proteomic approaches. The mutant strain P. putida UW4/AcdS-, lacking a functional 1-aminocyclopropane-1-carboxylic acid deaminase gene, was also assessed for its response to nickel stress.
Results: Two dimensional difference in-gel electrophoresis (DIGE) was used to detect significantly up- or down- regulated proteins (p 1.5) in P. putida in response to the presence of 2 mM Ni. Out of a total number of 1,702 proteins detected on the analytical gels for P. putida UW4, the expression levels of 82 (4.82%) proteins increased significantly while the expression of 81 (4.76%) proteins decreased significantly. Of 1,575 proteins detected on the analytical gels for P. putida UW4/AcdS-, the expression levels of 74 (4.70%) proteins increased and 51 (3.24%) proteins decreased significantly. Thirty-five proteins whose expression was altered were successfully identified by mass spectrometry and sequence comparisons with related species. Nineteen of the identified proteins were detected as differentially expressed in both wild-type and mutant expression profiles.
Conclusion: Functional assessment of proteins with significantly altered expression levels revealed several mechanisms thought to be involved in bacterial heavy metal detoxification, including general stress adaptation, anti-oxidative stress and heavy metal efflux proteins. This information may contribute to the development of plant growth-promoting bacteria mediated phytoremediation processes.
User PosterResearch Hughes C, Doble B, Xin L, Chen W, Shan P, Ma B, Lajoie G. SILAC Quantitation to a Depth of 3000 Proteins from a Double Knockout GSK-3 Line of Mouse Embryonic Stem Cells. ASMS 2009: MPB 056.
The use of SILAC permits the monitoring of pathways on a global level. However, without the ability to quantitate on large sample sets, there is a limitation on how much information can be extracted. As is shown here, PEAKS permits large scale analysis in a streamlined fashion, comparable to that of the TPP. The dataset has recently been expanded to >800000 MS/MS spectra from ~810 gigabytes of data. Further analysis on the global phosphorylation states of detected proteins using PEAKS is ongoing in an attempt to map modification changes as a result of the GSK-3 knockout.
User Poster Research Cao W, Ma M, Fu Q, Li L. HyPep: A New Strategy to Accelerate Peptide Discovery with A Combination of De Novo Sequencing and Homology Database Search. ASMS 2009.
PEAKS was used for automatic de novo sequencing. An in-house neuropeptide database containing 5825 entries was built for homology database searching. The sequence motif of a given peptide family was used for homology searching and the best-match database sequence can be assigned to the corresponding query sequence. A PO extract from Cancer borealis was used to evaluate this hybrid strategy. 17 possible peptides were screened from a total of 82 precursor ions and then 4 peptides were identified within minutes, while it typically requires a full-day work with manual sequencing alone. This hybrid method is efficient, accurate and more sensitive than BLAST.
User Published Research Speroni S, Rohayem J, Nenci S, Bonivento D, Robel I, Barthel J, Luzhkov VB, Coutard B, Canard B, Mattevi A. Structural and biochemical analysis of human pathogenic astrovirus serine protease at 2.0 A resolution. J Mol Biol. 2009 Apr 17;387(5):1137-52.
Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 A resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface beta-ribbons together with a "featureless" shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.
User Published Research Forné I, Agulleiro MJ, Asensio E, Abián J, Cerdà J. 2-D DIGE analysis of Senegalese sole (Solea senegalensis) testis proteome in wild-caught and hormone-treated F1 fish. Proteomics. 2009 Apr;9(8):2171-81.
In the farmed flatfish Senegalese sole, F1 males reared in captivity often show lower sperm production and fertilization capacity than wild-caught males. To gain insights into the molecular mechanisms that may be altered in the F1 testis, we used 2-D DIGE to compare the protein profiling of the testis of wild-caught males at the spermiation stage with that of F1 males showing different stages of germ cell development after hormone treatment in vivo. The abundance of 58 out of 1014 protein spots was found to differ significantly between the groups. De novo identification of these proteins by MS/MS revealed that proteins implicated in oxidoreductase activity, protein catabolism, formation of the zona pellucida receptor, cytoskeleton organization, and lipid binding and metabolism, were regulated in the F1 testes as germ cell development progressed. However, distinct isoforms or PTMs of some of these proteins, as well as of proteins involved in iron and glucose metabolism and ATP production, were expressed at lower levels in the testes of F1 males than in wild fish regardless of the hormone treatment. These results contribute to identifying proteins associated with spermatogenesis not previously described in teleosts, and suggest potential mechanisms that may be involved in the poor reproductive performance of Senegalese sole F1 males.
User Published Research Taddese S, Weiss AS, Jahreis G, Neubert RH, Schmelzer CE. In vitro degradation of human tropoelastin by MMP-12 and the generation of matrikines from domain 24. Matrix Biol. 2009 Mar;28(2):84-91.
Degradation of elastic fibers in tissues can result in the development of disorders that include aneurysms, atherosclerosis, and loss of skin elasticity. Tropoelastin is the precursor of the cross-linked elastin and its expression is triggered by elastin-degrading factors as a response to damage. Factors like UV radiation not only increase the expression of tropoelastin but also potent metalloelastases such as macrophage elastase (MMP-12). The development of elastin-degrading diseases, moreover, is a chronic process during which elastin and tropoelastin are repeatedly exposed to attacks by MMP-12. Hence, in this work we report the in vitro susceptibility of tropoelastin and the potential of MMP-12 to generate matrikines. This work provides evidence that tropoelastin is substantially and rapidly degraded by MMP-12 even at very dilute enzyme concentrations. MMP-12 cleaves at least 86 sites in tropoelastin. Analysis of the generated peptides revealed that some small peptides contained the motif GXXPG that may enable them to bind with the elastin binding protein (EBP). Furthermore, using synthesized peptides it was confirmed that several sites in the sequence encoded by exon 24 which contains repetitive units of biologically active VGVAPG domains are susceptible to attack by MMP-12, provided that the active subsites in MMP-12 (S(4) to S(4)') are occupied. Such cleavage events have lead to the generation of ligands that may bind to EBP.
User Published Research Viappiani S, Nicolescu AC, Holt A, Sawicki G, Crawford BD, León H, van Mulligen T, Schulz R. Activation and Modulation of 72kDa Matrix Metalloproteinase-2 by Peroxynitrite and Glutathione. Biochem Pharmacol. 2009 Mar 1;77(5):826-34.
Matrix metalloproteinase-2 (MMP-2) has emerged as a key protease in various pathologies associated with oxidative stress, including myocardial ischemia-reperfusion, heart failure or inflammation. Peroxynitrite (ONOO(-)), an important effector of oxidative stress, was reported to activate some full length MMP zymogens, particularly in the presence of glutathione (GSH), but whether this occurs for MMP-2 is unknown. Treating MMP-2 zymogen with ONOO(-) resulted in a concentration-dependent regulation of MMP-2, with 0.3-1 microM ONOO(-) increasing and 30-100 microM ONOO(-) attenuating enzyme activity. The enzyme's V(max) was also significantly increased by 1 microM ONOO(-). Comparable responses to ONOO(-) treatment were observed using the intracellular target of MMP-2, troponin I (TnI). GSH at 100 microM attenuated the effects of ONOO(-) on MMP-2. Mass spectrometry revealed that ONOO(-) can oxidize and, in the presence of GSH, S-glutathiolate the MMP-2 zymogen or a synthetic peptide containing the cysteine-switch motif in the enzyme's autoinhibitory domain. These results suggest that ONOO(-) and GSH can modulate the activity of 72 kDa MMP-2 by modifying the cysteine residue in the autoinhibitory domain of the zymogen, a process that may be relevant to pathophysiological conditions associated with increased oxidative stress.
User Published Research Navare A, Nouzova M, Noriega FG, Hernández-Martínez S, Menzel C, Fernández FM. On-chip solid-phase extraction pre-concentration/focusing substrates coupled to atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis. Rapid Commun Mass Spectrom. 2009 Feb;23(4):477-86.
Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP-MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP-MALDI suffers from less-than-optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal-to-noise ratio (S/N) gains observed when an on-chip dynamic pre-concentration/focusing approach is coupled to AP-MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP-MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200-900 for hydrophobic substrates, and 150-400 for weak cation-exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/microL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter-day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP-MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads.
User Published Research Chen T, Wu X, Chen Y, Li X, Huang M, Zheng M, Baluska F, Samaj J, Lin J. Combined proteomic and cytological analysis of Ca2+-calmodulin regulation in Picea meyeri pollen tube growth. Plant Physiol. 2009 Feb;149(2):1111-26.
Ca2+-calmodulin (Ca2+-CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca2+-CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca2+-CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca2+-CaM signaling rapidly induced an increase in extracellular Ca2+ influx, resulting in dramatically increased cytosolic Ca2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca2+-CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca2+-CaM in the regulation of tip growth machinery.
User Published Research Bringans S, Kendrick TS, Lui J, Lipscombe R. A comparative study of the accuracy of several de novo sequencing software packages for datasets derived by matrix-assisted laser desorption/ionisation and electrospray. Rapid Commun Mass Spectrom. 2008 Nov;22(21):3450-4.
The proteomic charactertisation of complex biological samples commonly utilises mass spectrometry techinques. Typically, the proteins are enzymatically digested and the resultant peptides analyzed via tandem mass spectrometry (MS/MS). This experimental MS/MS data is then compared to the theoretical fragmentation patterns of peptides derived from protein sequences contained within databases...
User Published Research Islam MA, Sturrock RN, Ekramoddoullah AK. A proteomics approach to identify proteins differentially expressed in Douglas-fir seedlings infected by Phellinus sulphurascens. J Proteomics. 2008 Oct 7;71(4):425-38.
We carried out a comparative proteomic study to explore the molecular mechanisms that underlie the defense response of Douglas-fir (DF, Pseudotsuga menziesii) to laminated root rot, a disease caused by Phellinus sulphurascens. 2-DE was conducted on proteins extracted from roots of laboratory-grown, young DF seedlings inoculated with P. sulphurascens. A total of 1303 proteins was detected in 7 dpi infected and uninfected root samples. Among these 1303 proteins, 277 showed differential expression that was statistically significant (p<0.05). Of these 277 proteins, 74 upregulated and 85 downregulated proteins showed at least a two-fold change from controls. Forty seven upregulated and 23 downregulated proteins were selected to be excised and analyzed using LC-MS/MS followed by peptide matching. Our results indicate that the major proteins differentially expressed in P. sulphurascens-infected DF seedlings include those in the following functional groups: disease/defense (27%), metabolism (16%), transcription factors (11%), signal transduction (10%), secondary metabolism (7%) and energy (4%). A number of additional proteins involved in cell structure (3%) and protein synthesis (3%) were also identified. By providing an initial database of candidate pathogenesis-related proteins for the DF-Phellinus sulphurascens pathosystem the results of this study will enable future detailed investigation of gene expression and function.
User Published Research Hoehenwarter W, van Dongen JT, Wienkoop S, Steinfath M, Hummel J, Erban A, Sulpice R, Regierer B, Kopka J, Geigenberger P, Weckwerth W. A Rapid Approach for Phenotype-Screening and Database Independent Detection of cSNP/Protein Polymorphism Using Mass Accuracy Precursor Alignment. Proteomics. 2008 Oct;8(20):4214-25.
The dynamics of a proteome can only be addressed with large-scale, high-throughput methods. To cope with the inherent complexity, techniques based on targeted quantification using proteotypic peptides are arising. This is an essential systems biology approach; however, for the exploratory discovery of unexpected markers, nontargeted detection of proteins, and protein modifications is indispensable. We present a rapid label-free shotgun proteomics approach that extracts relevant phenotype-specific peptide product ion spectra in an automated workflow without prior identification. These product ion spectra are subsequently sequenced with database search and de novo prediction algorithms. We analyzed six potato tuber cultivars grown on three plots of two geographically separated fields in Germany. For data mining about 1.5 million spectra from 107 analyses were aligned and statistically examined in approximately 1 day. Several cultivar-specific protein markers were detected. Based on de novo-sequencing a dominant protein polymorphism not detectable in the available EST-databases was assigned exclusively to a specific potato cultivar. The approach is applicable to organisms with unsequenced or incomplete genomes and to the automated extraction of relevant mass spectra that potentially cannot be identified by genome/EST-based search algorithms.
User Published Research Kang YJ, Stevenson AK, Yau PM, Kollmar R. Sparc Protein Is Required for Normal Growth of Zebrafish Otoliths. J Assoc Res Otolaryngol. 2008 December; 9(4): 436–451.
Otoliths and the homologous otoconia in the inner ear are essential for balance. Their morphogenesis is less understood than that of other biominerals, such as bone, and only a small number of their constituent proteins have been characterized. As a novel approach to identify unknown otolith proteins, we employed shotgun proteomics to analyze crude extracts from trout and catfish otoliths. We found three proteins that had not been associated previously with otolith or otoconia formation: ‘Secreted acidic cysteine rich glycoprotein’ (Sparc), an important bone protein that binds collagen and Ca2+; precerebellin-like protein, which contains a C1q domain and may associate with the collagenous otolin-1 during its assembly into a framework; and neuroserpin, a serine protease inhibitor that may regulate local protease activity during framework assembly. We then used the zebrafish to investigate whether Sparc plays a role in otolith morphogenesis. Immunodetection demonstrated that Sparc is a true constituent of otoliths. Knockdown of Sparc expression in morphant zebrafish resulted in four principal types of defective otoliths: smaller, extra and ectopic, missing and fused, or completely absent. Smaller size was the predominant phenotype and independent of the severity of otic-vesicle defects. These results suggested that Sparc is directly required for normal otolith growth.
User Published Research Martin-Visscher LA, van Belkum MJ, Garneau-Tsodikova S, Whittal RM, Zheng J, McMullen LM, Vederas JC. Isolation and characterization of carnocyclin a, a novel circular bacteriocin produced by Carnobacterium maltaromaticum UAL307. Appl Environ Microbiol. 2008 Aug;74(15):4756-63.
Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and alpha-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.
User Published Research Catusse J, Strub JM, Job C, Van Dorsselaer A, Job D. Proteome-wide characterization of sugarbeet seed vigor and its tissue specific expression. Proc Natl Acad Sci U S A. 2008 July 22; 105(29): 10262–10267.
Proteomic analysis of mature sugarbeet seeds led to the identification of 759 proteins and their specific tissue expression in root, cotyledons, and perisperm. In particular, the proteome of the perispermic storage tissue found in many seeds of the Caryophyllales is described here. The data allowed us to reconstruct in detail the metabolism of the seeds toward recapitulating facets of seed development and provided insights into complex behaviors such as germination. The seed appears to be well prepared to mobilize the major classes of reserves (the proteins, triglycerides, phytate, and starch) during germination, indicating that the preparation of the seed for germination is mainly achieved during its maturation on the mother plant. Furthermore, the data revealed several pathways that can contribute to seed vigor, an important agronomic trait defined as the potential to produce vigorous seedlings, such as glycine betaine accumulation in seeds. This study also identified several proteins that, to our knowledge, have not previously been described in seeds. For example, the data revealed that the sugarbeet seed can initiate translation either through the traditional cap-dependent mechanism or by a cap-independent process. The study of the tissue specificity of the seed proteome demonstrated a compartmentalization of metabolic activity between the roots, cotyledons, and perisperm, indicating a division of metabolic tasks between the various tissues. Furthermore, the perisperm, although it is known as a dead tissue, appears to be very active biochemically, playing multiple roles in distributing sugars and various metabolites to other tissues of the embryo.
User Published Research Fumagalli M, Dolcini L, Sala A, Stolk J, Fregonese L, Ferrari F, Viglio S, Luisetti M, Iadarola P. Proteomic analysis of exhaled breath condensate from single patients with pulmonary emphysema associated to alpha1-antitrypsin deficiency. J Proteomics. 2008 Jul 21;71(2):211-21.
The non-invasive character of exhaled breath (EBC) collection makes this fluid attractive for monitoring the respiratory tract by the measurement of various compounds. Because EBC is likely to reflect the composition of the airway-lining fluid, it can provide valuable information on possible disease states. Aim of our study was to apply proteomic technology to the study of EBC samples collected from single patients with pulmonary emphysema associated to alpha(1)-antitrypsin deficiency. The protein profiles from EBC of twenty patients and of twenty-five healthy individuals, used as controls, have been analyzed in parallel by a combination of 1-DE, 2-DE, micro-HPLC and MS. These sensitive techniques allowed to identify a number of cytokines and cytokeratins. Their level was found to be higher in patients than in controls.
User Published Research Taddese S, Weiss AS, Neubert RH, Schmelzer CE. Mapping of macrophage elastase cleavage sites in insoluble human skin elastin. Matrix Biol. 2008 Jun;27(5):420-8.
Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed.
User Published Research Pandhal J, Wright PC, Biggs CA. Proteomics with a pinch of salt: A cyanobacterial perspective. Saline Systems. 2008; 4: 1.
Cyanobacteria are ancient life forms and have adapted to a variety of extreme environments, including high salinity. Biochemical, physiological and genetic studies have contributed to uncovering their underlying survival mechanisms, and as recent studies demonstrate, proteomics has the potential to increase our overall understanding further. To date, most salt-related cyanobacterial proteomic studies have utilised gel electrophoresis with the model organism Synechocystis sp. PCC6803. Moreover, focus has been on 2–4% w/v NaCl concentrations within different cellular compartments. Under these conditions, Synechocystis sp. PCC6803 was found to respond and adapt to salt stress through synthesis of general and specific stress proteins, altering the protein composition of extracellular layers, and re-directing control of complex central intermediary pathways. Post-transcriptional control was also predicted through non-correlating transcript level data and identification of protein isoforms.In this paper, we also review technical developments with emphasis on improving the quality and quantity of proteomic data and overcoming the detrimental effects of salt on sample preparation and analysis. Developments in gel-free methods include protein and peptide fractionation workflows, which can increase coverage of the proteome (20% in Synechocystis sp. PCC6803). Quantitative techniques have also improved in accuracy, resulting in confidence in quantitation approaching or even surpassing that seen in transcriptomic techniques (better than 1.5-fold in differential expression). Furthermore, in vivo metabolic labelling and de novo protein sequencing software have improved the ability to apply proteomics to unsequenced environmental isolates. The example used in this review is a cyanobacterium isolated from a Saharan salt lake.
User Published Research Lacerda CM, Xin L, Rogers I, Reardon KF. Analysis of iTRAQ data using Mascot and Peaks quantification algorithms. Brief Funct Genomic Proteomic. 2008 Mar;7(2):119-26.
The field of proteomics has been developing rapidly toward quantification of proteins. Despite the variety of experimental techniques available for peptide and protein labelling, there are few commercially available analytical tools with the ability to interpret data from any mass spectrometer. In this study, we compare two software packages, Mascot and Peaks, for the analysis of iTRAQ data from ESI-Q/TOF mass spectrometry. In the case of a six-protein mixture combined in a known proportion, the output of the Peaks algorithm deviated from the correct result by 14% on average, while the error of the Mascot quantification was nearly 200%.When the software were used to analyze iTRAQ data from a complex protein sample, the quantification results agreed within 20% for only 26% of the quantified proteins, showing significant differences in the two quantification algorithms. This comparison and analysis revealed major intricacies in peptide and protein quantification that must be taken into consideration for software development.
User Published Research Hatano N, Hamada T. Proteome Analysis of Pitcher Fluid of the Carnivorous Plant Nepenthes Alata. J Proteome Res. 2008 Feb;7(2):809-16.
The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.
User Published Research Chiang YR, Ismail W, Heintz D, Schaeffer C, Van Dorsselaer A, Fuchs G. Study of anoxic and oxic cholesterol metabolism by Sterolibacterium denitrificans. J Bacteriol. 2008 Feb;190(3):905-14.
The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1S(T). The second enzyme of the proposed pathway, cholest-4-en-3-one-Delta1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.
User Published Research Yeo TH, Ho ML, Loke WK. Development of a Liquid Chromatography-Multiple Reaction Monitoring Procedure for Concurrent Verification of Exposure to Different Forms of Mustard Agents. J Anal Toxicol. 2008 Jan-Feb;32(1):51-6.
A novel liquid chromatography-multiple reaction monitoring (LC-MRM) procedure has been developed for retrospective diagnosis of exposure to different forms of mustard agents. This concise method is able to validate prior exposure to nitrogen mustards (HN-1, HN-2, and HN-3) or sulfur mustard (HD) in a single run, which significantly reduces analysis time compared to separate runs to screen for different mustards' biomarkers based on tandem mass spectrometry. Belonging to one of the more toxic classes of chemical warfare agents, these potent vesicants bind covalently to the cysteine-34 residue of human serum albumin. This results in the formation of stable adducts whose identities were confirmed by a de novo sequencing bioinformatics software package. Our developed technique tracks these albumin-derived adduct biomarkers in blood samples which persist in vitro following exposure, enabling a detection limit of 200 nM of HN-1, 100 nM of HN-2, 200 nM of HN-3, or 50 nM of HD in human blood. The CWA-adducts formed in blood samples can be conveniently and sensitively analyzed by this MRM technique to allow rapid and reliable screening.
User Published Research Alonso-Hearn M, Patel D, Danelishvili L, Meunier-Goddik L, Bermudez LE. The Mycobacterium avium subsp. paratuberculosis MAP3464 gene encodes an oxidoreductase involved in invasion of bovine epithelial cells through the activation of host cell Cdc42. Infect Immun. 2008 Jan;76(1):170-8.
Mycobacterium avium subsp. paratuberculosis infection of cattle takes place through the intestinal mucosa. To identify M. avium subsp. paratuberculosis genes associated with the invasion of bovine epithelial cells in vitro, we screened a library of transposon mutants. Several mutants of M. avium subsp. paratuberculosis were identified which invaded Madin-Darby bovine kidney (MDBK) epithelial cells less efficiently than wild-type (wt) M. avium subsp. paratuberculosis. The deltaOx mutant had the transposon located in the MAP3464 gene, a putative oxidoreductase gene whose expression is upregulated upon bacterial contact with MDBK cells. Complete restoration of invasion comparable to that for the wt bacterium was achieved by introducing a copy of the complete oxidoreductase operon into the deltaOx mutant. Immunoprecipitation and Western blot analysis indicated that wt M. avium subsp. paratuberculosis activates Cdc42 and RhoA pathways of internalization 15 and 60 min after infection of the host cell, respectively. The deltaOx mutant, however, failed to activate the Cdc42 pathway. To determine whether an M. avium subsp. paratuberculosis protein delivered to the host cell mediates the entry of the wt bacterium by activation of the Cdc42 pathway, affinity precipitation of active Cdc42 from MDBK-infected cells followed by mass spectrometry was carried out. We identified a 17-amino-acid bacterial peptide associated with the Cdc42 of cells infected with wt M. avium subsp. paratuberculosis but not with the deltaOx mutant. The sequence of the peptide matches MAP3985c, a hypothetical protein, possibly functioning as a putative Cdc42 effector. These findings reveal a novel signaling pathway activated during M. avium subsp. paratuberculosis entry that links the product of MAP3464 gene to activation of Cdc42 in the host cell.
User Published Research Führs H, Hartwig M, Molina LE, Heintz D, Van Dorsselaer A, Braun HP, Horst WJ. Early manganese-toxicity response in Vigna unguiculata L.--a proteomic and transcriptomic study. Proteomics. 2008 Jan;8(1):149-59.
The apoplast is known to play a predominant role in the expression of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.) leaves. To unravel early Mn-toxicity responses after 1-3 days Mn treatment also in the leaf symplast, we studied the symplastic reactions induced by Mn in two cultivars differing in Mn tolerance on a total cellular level. Comparative proteome analyses of plants exposed to low or high Mn allowed to identify proteins specifically affected by Mn, particularly in the Mn-sensitive cowpea cultivar. These proteins are involved in CO(2) fixation, stabilization of the Mn cluster of the photosystem II, pathogenesis-response reactions and protein degradation. Chloroplastic proteins important for CO(2) fixation and photosynthesis were of lower abundance upon Mn stress suggesting scavenging of metabolic energy for a specific stress response. Transcriptome analyses supported these findings, but additionally revealed an upregulation of genes involved in signal transduction only in the Mn-sensitive cultivar. In conclusion, a coordinated interplay of apoplastic and symplastic reactions seems to be important during the Mn-stress response in cowpea.
User Published Research Portelius E, Tran AJ, Andreasson U, Persson R, Brinkmalm G, Zetterberg H, Blennow K, Westman-Brinkmalm A. Characterization of Amyloid Beta Peptides in Cerebrospinal Fluid by an Automated Immunoprecipitation Procedure Followed by Mass Spectrometry. J Proteome Res. 2007 Nov;6(11):4433-9.
Pathogenic events in Alzheimer's disease are believed to involve an imbalance between the production and clearance of the neurotoxic 42 amino acid form of the beta-amyloid peptide (Abeta1-42). Although much is known about the production of Abeta1-42, many questions remain about its degradation. Here, we describe an optimized automated immunoprecipitation mass spectrometry method that enables accurate and rapid monitoring of the major Abeta isoforms in cerebrospinal fluid. Furthermore, we describe a technique of antibody immobilization, minimizing background signals. The identities of these Abeta products were confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoflow liquid chromatography and tandem mass spectrometry with a hybrid linear trap Fourier transform ion cyclotron resonance mass spectrometer. Finally, we report the finding of two novel Abeta peptides (Abeta2-17 and Abeta3-17).
User Published Research Bertile F, Robert F, Delval-Dubois V, Sanglier S, Schaeffer C, Van Dorsselaer A. Endogenous plasma Peptide detection and identification in the rat by a combination of fractionation methods and mass spectrometry. Biomark Insights. 2007 Oct 9;2:385-401.
Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%-35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/muL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10(-6)% of total proteins, by weight. Signal peptide recovery ranged between 5%-100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction.
User Published Research Schmelzer CE, Schöps R, Reynell L, Ulbrich-Hofmann R, Neubert RH, Raith K. Peptic Digestion of Beta-Casein. Time Course and Fate of Possible Bioactive Peptides. J Chromatogr A. 2007 Sep 28;1166(1-2):108-15.
Numerous peptides obtained by enzymatic digestion of food proteins have been reported to exhibit biological activities. In this study, the focus was placed on peptides of beta-casein from bovine milk after a gastro-analogous in vitro digestion with pepsin, a protease with broad specificity. In order to study the time course of the digestion, the process was stopped after specific times and the samples were subjected to HPLC separation followed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and nanoelectrospray (nanoESI) quadrupole time-of-flight (qTOF) mass spectrometry. A combined sequencing approach using de novo interpretation and databases was employed. Overall, 100% of the beta-casein sequence was covered by identifying 125 peptides of 4-84 residues in length, including 3 phosphorylated species. The results show that the peptic hydrolysis starts at the C-terminus of the protein. The release of known bioactive peptides from beta-casein following the peptic digestion under simulated gastric conditions is unlikely with a few exceptions. Furthermore, an amino acid variation was found, providing evidence for the existence of an additional genetic variant of beta-casein.
User Published Research Liu T, Belov ME, Jaitly N, Qian WJ, Smith RD. Accurate Mass Measurements in Proteomics. Chem Rev. 2007 August; 107(8): 3621–3653.
Herein, we review the presently most important and promising topics in proteomics applying accurate mass measurements rather than the broader area of proteomics, which has been discussed and summarized in many excellent reviews.6-16 The two general approaches to MS-based proteomics and a brief discussion on the need for accurate mass measurements complete this introduction prior to reviewing high-resolution MS instrumentation and methods that provide high mass measurement accuracy (MMA), improvements in proteomics applications applying accurate mass measurements, and developments in bioinformatics that utilize high mass accuracy data to enable new data analysis strategies.
User Published Research St Pierre L, Birrell GW, Earl ST, Wallis TP, Gorman JJ, de Jersey J, Masci PP, Lavin MF. Diversity of toxic components from the venom of the evolutionarily distinct black whip snake, Demansia vestigiata. J Proteome Res. 2007 Aug;6(8):3093-107.
Included among the more than 300 species of elapid snakes worldwide is the Australian genus Demansia, or whip snakes. Despite evidence to suggest adverse clinical outcomes from envenomation by these snakes, together with confusion on their true phylogenetic relationship to other Australian elapids, not a single toxin sequence has previously been reported from the venom of a Demansia species. We describe here a combined proteomic and transcriptomic approach characterizing the venom from the black whip snake, Demansia vestigiata. A total of 13 distinct toxin families were identified, including homologues of all of the major toxic components previously reported from the venom of other Australian elapids, such as factor X-like prothrombin activators, neurotoxins, phospholipases, cysteine rich secretory proteins, textilinin-like molecules, nerve growth factors, l-amino acid oxidases, vespryns, 5' nucleotidases, metalloproteinases, and C-type lectins as well as a novel dipeptidyl peptidase family. Phylogenetic analysis of these sequences revealed an early evolutionary split of the black whip snake from all other characterized Australian snakes, with a low degree of sequence identity between D. vestigiata and the other snakes, across all toxin families. The results of this study have important implications not only for the further characterization of venom from whip snakes, but also for our understanding of the evolutionary relationship of Australian snake species.
User Published Research Domanski D, Helbing CC. Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin. BMC Dev Biol. 2007 Aug 6;7:94.
Background: Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action.
Results: Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport.
Conclusion: We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study identifies proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole tail regression prior to overt remodeling of the tail. In particular, we have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone.
User Published Research Tannu NS, Hemby SE. De novo protein sequence analysis of Macaca mulatta. BMC Genomics. 2007 Aug 8;8:270.
Background: Macaca mulatta is one of the most utilized non-human primate species in biomedical research offering unique behavioral, neuroanatomical, and neurobiochemcial similarities to humans. This makes it a unique organism to model various diseases such as psychiatric and neurodegenerative illnesses while also providing insight into the complexities of the primate brain. A major obstacle in utilizing rhesus monkey models for human disease is the paucity of protein annotations for this species (~42,000 protein annotations) compared to 330,210 protein annotations for humans. The lack of available information limits the use of rhesus monkey for proteomic scale studies which rely heavily on database searches for protein identification. While characterization of proteins of interest from Macaca mulatta using the standard database search engines (e.g., MASCOT) can be accomplished, searches must be performed using a 'broad species database' which does not provide optimal confidence in protein annotation. Therefore, it becomes necessary to determine partial or complete amino acid sequences using either manual or automated de novo peptide sequence analysis methods.
Results: The recently popularized MALDI-TOF-TOF mass spectrometer yields a complex MS/MS fragmentation pattern difficult to characterize by manual de novo sequencing method on a proteomics scale. Therefore, PEAKS assisted de novo sequencing was performed on nucleus accumbens cytosolic proteins from Macaca mulatta. The most abundant peptide fragments 'b-ions and y-ions', the less abundant peptide fragments 'a-ions' as well as the immonium ions were utilized to develop confident and complete peptide sequences de novo from MS/MS spectra. The generated sequences were used to perform homology searches to characterize the protein identification.
Conclusion: The current study validates a robust method to confidently characterize the proteins from an incomplete sequence database of Macaca mulatta, using the PEAKS de novo sequencing software, facilitating the use of this animal model in various neuroproteomics studies.
User Published Research Danelishvili L, Wu M, Stang B, Harriff M, Cirillo SL, Cirillo JD, Bildfell R, Arbogast B, Bermudez LE. Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection. Proc Natl Acad Sci U S A. 2007 Jun 26;104(26):11038-43.
The ability to infect macrophages is a common characteristic shared among many mycobacterial species. Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium kansasii enter macrophages, using the complement receptors CR1, CR3, CR4, and the mannose receptor. To identify M. avium genes and host cell pathways involved in the bacterial uptake by macrophages, we screened a M. avium transposon mutant library for the inability to enter macrophages. Uptake-impaired clones were selected. Sequence of six M. avium clones identified one gene involved in glycopeptidolipid biosynthesis, one gene encoding the conserved membrane protein homologue to the M. avium subsp. paratuberculosis MAP2446c gene and four others belonging to the same region of the chromosome. Analysis of the chromosome region revealed a pathogenicity island inserted between two tRNA sequences with 58% of G+C content versus 69% in the M. avium genome. The region is unique for M. avium and is not present in M. tuberculosis or M. paratuberculosis. Although the mutants did not differ from the WT bacterium regarding the binding to macrophage cell membrane, analysis of macrophage proteins after 1 h infection revealed a deficiency in the mutant to phosphorylate certain proteins on uptake. To understand M. avium interaction with two evolutionarily distinct hosts, the mutants were evaluated for Acanthamoeba castellanii invasion. The defect in the ability of the mutants to invade both cells was highly similar, suggesting that M. avium might have evolved mechanisms that are used to enter amoebas and human macrophages.
User Published Research Chen YT, Tsao CY, Li JM, Tsai CY, Chiu SF, Tseng TL. Large-scale protein identification of human urine proteome by multi-dimensional LC and MS/MS. Proteomics Clin Appl. 2007 Jun;1(6):577-87.
Urine is a human specimen that is easily obtained non-invasively for clinical diagnosis. We attempted to enhance the resolution of current human urine proteomes and construct a comprehensive reference database for advanced studies, such as the discovery of biomarkers for renal diseases. Multi-dimensional LC-MS/MS was coupled with de novo sequencing and database matching. The proposed approach improved the identification of not only the proteins, but also the post-translational sites of urinary proteins. We identified 165, 200 and 259 unique gene products in the urine proteomes from males, females and pregnant women, respectively. When all of the results were combined and the redundancies removed, a total of 1095 distinct peptides were identified. Of these, 1016 peptides were associated with 334 unique gene products. In this study, over 100 gene products, including some disease-related proteins, were detected in urine for the first time by proteomic approaches. Various proteins with novel post-translational hydroxylation were identified using the MASCOT program and de novo sequencing. All proteins with peptide information were summarized into a comprehensive urine protein database. We believe that this comprehensive urine proteome database will assist in the identification of urinary proteins/polypeptides whose spectra are difficult to interpret in the discovery of urinary biomarkers.
User Published Research Pereira-Medrano AG, Margesin R, Wright PC. Proteomic characterization of the psychrophile Pedobacter cryoconitis based on both 15N metabolic labeling and de novo sequencing. ASMS 2007 Poster presentation.
Characterizing the proteome of an unsequenced psychrophile grown with different carbon sources and under temperature extremes employing 15N metabolic labeling, 2DGE, tandem mass spectrometry, N-constrained ortholog searching, and de novo sequencing.
User Poster Research Rogers I, Scigelova M, Woffendin G. Optimizing Data Acquisition for Automated de novo Sequencing. ASMS 2007.
De novo sequencing enables identification of peptides and proteins from unsequenced genomes or validation of the results of a database search. To be of practical use this process must be automated, with a throughput matching that of data acquisition. The LTQ Orbitrap delivers routine mass measurements with deviations of less than 3 ppm (external calibration). In the context of proteomics experiments measuring the precursor ion highly accurately means fewer false positive identifications. There is, however, no clear consensus regarding the benefit of MS/MS mass accuracy. This is because the accurate mass detection in the Orbitrap analyzer takes longer than the fragment detection in a linear ion trap, resulting in potentially less peptides being fragmented and identified. Also, the LTQ Orbitrap can fragment peptides in the linear ion trap or in the C-trap, each method being characterized by particular spectra qualities. We performed a detailed comparison of data acquisition methods on LTQ Orbitrap with respect to their suitability for automated de novo sequencing with PEAKS Studio 4.2 software. As this package combines de novo sequencing with BLAST searches in databases we were also interested in indications of amino acid substitutions or unexpected modifications.
User Poster Research Thalassinos K, Efstathiou G, Slade SE, Scrivens JH. An Objective Organism-Based Evaluation of Tandem Mass Spectrometric Data Obtained from Proteomic Studies. ASMS 2007.
An oligomer-segmented organism specific database approach has been developed which, when combined with de-novo sequencing information, provides objective evaluation of the information content of peptide MS/MS spectra. The majority of one-hit-wonder identifications by traditional database searching were found to be false positives, but some were confirmed, and several new proteins discovered.
User Published Research Birrell GW, Earl ST, Wallis TP, Masci PP, de Jersey J, Gorman JJ, Lavin MF. The diversity of bioactive proteins in Australian snake venoms. Mol Cell Proteomics. 2007 Jun;6(6):973-86.
Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from 10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.
User Published Research Kanazawa M, Anyoji H, Ogiwara A, Nagashima U. De novo peptide sequencing using ion peak intensity and amino acid cleavage intensity ratio. Bioinformatics. 2007 May 1;23(9):1068-72.
Motivation: Peptide-sequencing methods by mass spectrum use the following two approaches: database searching and de novo sequencing. The database-searching approach is convenient; however, in cases wherein the corresponding sequences are not included in the databases, the exact identification is difficult. On the other hand, in the case of de novo sequencing, no preliminary information is necessary; however, continuous amino acid sequence peaks and the differentiation of these peaks are required. It is, however, very difficult to obtain and differentiate the peaks of all amino acids by using an actual spectrum. We propose a novel de novo sequencing approach using not only mass-to-charge ratio but also ion peak intensity and amino acid cleavage intensity ratio (CIR). Results: Our method compensates for any undetectable amino acid peak intervals by estimating the amino acid set and the probability of peak expression based on amino acid CIR. It provides more accurate identification of sequences than the existing methods, by which it is usually difficult to sequence.
User Published Research Tolleter D, Jaquinod M, Mangavel C, Passirani C, Saulnier P, Manon S, Teyssier E, Payet N, Avelange-Macherel MH, Macherel D. Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation. Plant Cell. 2007 May;19(5):1580-9.
Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.
User Published Research Nagalla SR, Canick JA, Jacob T, Schneider KA, Reddy AP, Thomas A, Dasari S, Lu X, Lapidus JA, Lambert-Messerlian GM, Gravett MG, Roberts CT Jr, Luthy D, Malone FD, D'Alton ME. Proteomic analysis of maternal serum in down syndrome: identification of novel protein biomarkers. J Proteome Res. 2007 Apr;6(4):1245-57.
Down syndrome (DS) is the most prevalent chromosomal disorder, accounting for significant morbidity and mortality. Definitive diagnosis requires invasive amniocentesis, and current maternal serum-based testing requires a false-positive rate of about 5% to detect 85% of affected pregnancies. We have performed a comprehensive proteomic analysis to identify potential serum biomarkers to detect DS. First- and second-trimester maternal serum samples of DS and gestational age-matched controls were analyzed using multiple, complementary proteomic approaches, including fluorescence 2-dimensional gel electrophoresis (2D-DIGE), 2-dimensional liquid chromatography-chromatofocusing (2D-CF), multidimensional protein identification technology (MudPIT; LC/LC-MS/MS), and MALDI-TOF-MS peptide profiling. In total, 28 and 26 proteins were differentially present in first- and second-trimester samples, respectively. Of these, 19 were specific for the first trimester and 16 for the second trimester, and 10 were differentially present in both trimesters. Analysis of MALDI-TOF-MS peptide profiles with pattern-recognition software also discriminated between DS and controls in both trimesters, with an average recognition capability approaching 96%. A majority of the biomarkers identified are serum glycoproteins that may play a role in cellular differentiation and growth of fetus. Further characterization and quantification of these markers in a larger cohort of subjects may provide the basis for new tests for improved DS screening.
User Published Research Lacerda CM, Choe LH, Reardon KF. Metaproteomic analysis of a bacterial community response to cadmium exposure. J Proteome Res. 2007 Mar;6(3):1145-52.
Microbial communities are of great environmental, medical, and industrial significance. To date, biomolecular methods to study communities have focused on identifying species, with limited capabilities to reveal functions. Proteomics has the potential to yield functional information about these communities, but the application of proteomic methods to complex mixtures of unsequenced organisms is in its infancy. In this study, 2DE, MALDI-TOF/TOF MS, and de novo peptide sequencing were used for the separation and identification of proteins differentially expressed over time following exposure of a bacterial community to an inhibitory level of cadmium. Significant community proteome responses after 0.25, 1, 2, and 3 h of exposure to cadmium were observed, with more than 100 protein expression changes detected at each time point. Several temporal responses were observed, and the most common expression pattern was immediate up- or down-regulation within 15 min of shock followed by maintenance of that level. More than 100 unique differentially expressed proteins were identified through database searching and de novo sequencing. Proteins of importance in the cadmium shock included ATPases, oxidoreductases, and transport proteins. The ability of proteomics to detect the differential regulation of these proteins even during short cadmium exposures shows that it is a powerful tool in explaining cellular mechanisms for a mixed culture. This is the first report of the large-scale identification of proteins involved in the dynamic response of a community of unsequenced bacteria using de novo sequencing.
User Published Research Tunsjø HS, Paulsen SM, Mikkelsen H, L'abée-Lund TM, Skjerve E, Sørum H. Adaptive response to environmental changes in the fish pathogen Moritella viscosa. Res Microbiol. 2007 Apr;158(3):244-50.
The marine psychrophilic bacterium Moritella viscosa is the causative agent of winter ulcer in farmed Atlantic salmon and cod. In this study, the growth requirements of the pathogen were established. The effects of changes in salinity and temperature on growth, surface features and proteomic regulation were also investigated. The genome of this bacterium has not yet been sequenced; therefore, comparative two-dimensional gel electrophoresis (2-DE) was used, coupled with high performance tandem mass spectrometry (MS/MS), to perform cross-species protein identification. Results from this study establish that M. viscosa is a true marine psychrophilic bacterium capable of surviving and proliferating in an oligotrophic and cold environment. Low temperature combined with 3-4% NaCl resulted in significantly higher cell yields and stability compared to high temperature and 1% NaCl. Nine cytoplasmic proteins were shown to be regulated by temperature and 12 by salinity. Several of the regulated proteins indicated a stressful situation at 15 degrees C compared to 4 degrees C, consistent with the growth characteristics observed. Furthermore, temperature and salinity were demonstrated to be important determinants of motility and viscosity of M. viscosa.
User Published Research Jaquinod M, Villiers F, Kieffer-Jaquinod S, Hugouvieux V, Bruley C, Garin J, Bourguignon J. A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture. Mol Cell Proteomics. 2007 Mar;6(3):394-412.
To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker alpha-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs.
User Published Research Wagner, RE. Mugnaini, S. Sniezko, R. Hardie, D. Poulis, B. Nepi, M. Pacini, E. von Aderkas, P. Proteomic Evaluation of Gymnosperm Pollination Drop Proteins Indicates Highly Conserved and Complex Biological Functions. Sexual Plant Reproduction 2007, Volume 20, Number 4, 181-189.
The pollination droplet is a highly conservative pollination mechanism that is observed in all major gymnosperm taxa. Proteomics analysis of the pollination drops was carried out on four gymnosperm species: Juniperus communis (common juniper), Juniperus oxycedrus (prickly juniper), Chamaecyparis lawsoniana (Port Orford cedar), and Welwitschia mirabilis. Pollination drop proteins were purified by SDS-PAGE, and the most abundant proteins were analyzed by mass spectrometry and sequenced. Based on BLAST searching of combined amino acid sequences, the following proteins were identified in the following species: an 83-kDa subtilisin-like proteinase, a 62-kDa glycosyl hydrolase, a 47.5-kDa glucan 1,3-ß-glucosidase precursor, a 30-kDa chitinase, and a 25-kDa thaumatin-like protein were identified in J. communis; a 30-kDa chitinase, a 25-kDa thaumatin-like protein, and a 32.5-kDa glucanase-like protein were identified in J. oxycedrus; an 83-kDa subtilisin-like proteinase, a 62-kDa ß-d-glucan exohydrolase, a 47.5-kDa glucan 1,3-ß-glucosidase, and two 25-kDa thaumatin-like proteins were identified in C. lawsoniana, and a 25-kDa chitinase was identified in W. mirabilis. Based on protein identifications, there is strong evidence that the pollination drop functions in both pathogen defense and pollen development. The discovery of similarities in terms of peptide sequence and protein identifications indicates that ovular secretions are functionally conservative, and that they are essential to reproductive success.
User Published Research Raeder IL, Paulsen SM, Smalås AO, Willassen NP. Effect of fish skin mucus on the soluble proteome of Vibrio salmonicida analysed by 2-D gel electrophoresis and tandem mass spectrometry. Microb Pathog. 2007 Jan;42(1):36-45.
Vibrio salmonicida is the causative agent of cold-water vibriosis in farmed marine fish species. Adherence of pathogenic bacteria to mucosal surfaces is considered to be the first steps in the infective processes, and proteins involved are regarded as virulence factors. The global protein expression profile of V. salmonicida, grown with and without the presence of fish skin mucus in the synthetic media, was compared. Increased levels of proteins involved in motility, oxidative stress responses, and general stress responses were demonstrated as an effect of growth in the presence of mucus compared to non-mucus containing media. Enhanced levels of the flagellar proteins FlaC, FlaD and FlaE indicate increased motility capacity, while enhanced levels of the heat shock protein DnaK and the chaperonin GroEL indicate a general stress response. In addition, we observed that peroxidases, TPx.Grx and AhpC, involved in the oxidative stress responses, were induced by mucus proteins. The addition of mucus to the culture medium did not significantly alter the growth rate of V. salmonicida. An analysis of mucus proteins suggests that the mucus layer harbours a protein species that potentially possesses catalytic activity against DNA, and a protein with iron chelating activity. This study represents the first V. salmonicida proteomic analysis, and provides specific insight into the proteins necessary for the bacteria to challenge the skin mucus barrier of the fish.
User Published Research Snijders AP, de Koning B, Wright PC. Relative Quantification of Proteins Across the Species Boundary Through the Use of Shared Peptides. J Proteome Res. 2007 Jan;6(1):97-104.
We show that shared peptides of proteins that are encoded in different species are suitable for cross-species relative protein quantification. A 14N-containing proteome from the thermoacidophilic archaeon Sulfolobus tokodaii was mixed with a 15N-labeled proteome from Sulfolobus solfataricus. Using three shared peptides per protein, the relative abundance of six orthologous proteins was calculated. Observed standard deviations were approximately 10%, indicating that the trypsin accessibility to cleavage sites was not altered in the orthologs. The abundance ratios of the and subunits of the Thermosome were 0.64 and 1.24 in Sulfolobus tokodaii compared to Sulfolobus solfataricus, suggesting a different stoichiometry of the complex in both species. In addition, an in silico study was performed on the occurrence of shared peptides. Inter- and intra-species peptide redundancy was investigated in the model organisms Homo sapiens, Mus musculus, Escherichia coli K12, Escherichia coli O157:H7, S. solfataricus, and S. tokodaii. M. musculus and H. sapiens share 30-50% of all peptides (6-15 residues). Moreover, approximately one-third of all proteins shared > or = 40% of their peptides with at least one other protein in the related species, thus offering strong potential for cross-species relative protein quantification. Conversely, approximately 40% of all peptides (6-15 residues) encoded in H. sapiens are encoded multiple times and therefore complicate identification and quantification.
User Published Research Pevtsov S, Fedulova I, Mirzaei H, Buck C, Zhang X. Performance evaluation of existing de novo sequencing algorithms. J Proteome Res. 2006 Nov;5(11):3018-28.
Two methods have been developed for protein identification from tandem mass spectra: database searching and de novo sequencing. De novo sequencing identifies peptide directly from tandem mass spectra. Among many proposed algorithms, we evaluated the performance of the five de novo sequencing algorithms, AUDENS, Lutefisk, NovoHMM, PepNovo, and PEAKS. Our evaluation methods are based on calculation of relative sequence distance (RSD), algorithm sensitivity, and spectrum quality. We found that de novo sequencing algorithms have different performance in analyzing QSTAR and LCQ mass spectrometer data, but in general, perform better in analyzing QSTAR data than LCQ data. For the QSTAR data, the performance order of the five algorithms is PEAKS > Lutefisk, PepNovo > AUDENS, NovoHMM. The performance of PEAKS, Lutefisk, and PepNovo strongly depends on the spectrum quality and increases with an increase of spectrum quality. However, AUDENS and NovoHMM are not sensitive to the spectrum quality. Compared with other four algorithms, PEAKS has the best sensitivity and also has the best performance in the entire range of spectrum quality. For the LCQ data, the performance order is NovoHMM > PepNovo, PEAKS > Lutefisk > AUDENS. NovoHMM has the best sensitivity, and its performance is the best in the entire range of spectrum quality. But the overall performance of NovoHMM is not significantly different from the performance of PEAKS and PepNovo. AUDENS does not give a good performance in analyzing either QSTAR and LCQ data.
User Published Research Wilson, JJ. Brodbelt, JS. Infrared Multiphoton Dissociation for Enhanced De Novo Sequence Interpretation of N-terminal Sulfonated Peptides in a Quadrupole Ion Trap. Anal Chem. 2006 Oct 1;78(19):6855-62.
Infrared multiphoton dissociation (IRMPD) of N-terminal sulfonated peptides improves de novo sequencing capabilities in a quadrupole ion trap mass spectrometer. Not only does IRMPD promote highly efficient dissociation of the N-terminal sulfonated peptides but also the entire series of y ions down to the y(1) fragment may be detected due to alleviation of the low-mass cutoff problem associated with conventional collisional activated dissociation (CAD) methods in a quadrupole ion trap. Commercial de novo sequencing software was applied for the interpretation of CAD and IRMPD MS/MS spectra collected for seven unmodified peptides and the corresponding N-terminal sulfonated species. In most cases, the additional information obtained by N-terminal sulfonation in combination with IRMPD provided significant improvements in sequence identification. The software sequence tag results were combined with a commercial database searching algorithm to interpret sequence information of a tryptic digest on alpha-casein s1. Energy-variable CAD studies confirmed a 30-40% reduction in the critical energies of the N-terminal sulfonated peptides relative to unmodified peptides. This reduction in dissociation energy facilitates IRMPD in a quadrupole ion trap.
User Published Research Lamont RJ, Meila M, Xia Q, Hackett M. Mass spectrometry-based proteomics and its application to studies of Porphyromonas gingivalis invasion and pathogenicity. Infect Disord Drug Targets. 2006 September; 6(3): 311–325.
Porphyromonas gingivalis is a Gram-negative anaerobe that populates the subgingival crevice of the mouth. It is known to undergo a transition from its commensal status in healthy individuals to a highly invasive intracellular pathogen in human patients suffering from periodontal disease, where it is often the dominant species of pathogenic bacteria. The application of mass spectrometry-based proteomics to the study of P. gingivalis interactions with model host cell systems, invasion and pathogenicity is reviewed. These studies have evolved from qualitative identifications of small numbers of secreted proteins, using traditional gel-based methods, to quantitative whole cell proteomic studies using multiple dimension capillary HPLC coupled with linear ion trap mass spectrometry. It has become possible to generate a differential readout of protein expression change over the entire P. gingivalis proteome, in a manner analogous to whole genome mRNA arrays. Different strategies have been employed for generating protein level expression ratios from mass spectrometry data, including stable isotope metabolic labeling and most recently, spectral counting methods. A global view of changes in protein modification status remains elusive due to the limitations of existing computational tools for database searching and data mining. Such a view would be desirable for purposes of making global assessments of changes in gene regulation in response to host interactions during the course of adhesion, invasion and internalization. With a complete data matrix consisting of changes in transcription, protein abundance and protein modification during the course of invasion, the search for new protein drug targets would benefit from a more comprehensive understanding of these processes than what could be achieved prior to the advent of systems biology.
User Published Research Tian G, Huang MC, Parvari R, Diaz GA, Cowan NJ. Cryptic out-of-frame translational initiation of TBCE rescues tubulin formation in compound heterozygous HRD. Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13491-6.
Microtubules are indispensable dynamic structures that contribute to many essential biological functions. Assembly of the native alpha/beta tubulin heterodimer, the subunit that polymerizes to form microtubules, requires the participation of several molecular chaperones, namely prefoldin, the cytosolic chaperonin CCT, and a series of five tubulin-specific chaperones termed cofactors A-E (TBCA-E). Among these, TBCC, TBCD, and TBCE are essential in higher eukaryotes; they function together as a multimolecular machine that assembles quasinative CCT-generated alpha- and beta-tubulin polypeptides into new heterodimers. Deletion and truncation mutations in the gene encoding TBCE have been shown to cause the rare autosomal recessive syndrome known as HRD, a devastating disorder characterized by congenital hypoparathyroidism, mental retardation, facial dysmorphism, and extreme growth failure. Here we identify cryptic translational initiation at each of three out-of-frame AUG codons upstream of the genetic lesion as a unique mechanism that rescues a mutant HRD allele by producing a functional TBCE protein. Our data explain how afflicted individuals, who would otherwise lack the capacity to make functional TBCE, can survive and point to a limiting capacity to fold tubulin heterodimers de novo as a contributing factor to disease pathogenesis.
User Published Research Winkelmann T, Heintz D, Van Dorsselaer A, Serek M, Braun HP. Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum Mill. reveal new insights into seed and germination physiology. Planta. 2006 Aug;224(3):508-19.
In the horticulturally important ornamental species Cyclamen persicum Mill., somatic embryogenesis is an efficient vegetative propagation method and the development of artificial seeds is an ultimate aim. This study aims at a systematic comparison of the proteomes of zygotic embryos, somatic embryos grown in liquid medium containing 30 or 60 g l(-1) sucrose, germinating embryos of both types and endosperm in order to obtain novel insights into seed and germination physiology. Using high resolution two-dimensional isoelectric focussing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D IEF/SDS PAGE), 74% of the proteins expressed in zygotic embryos were found in similar abundance in somatic embryos grown in 60 g l(-1) sucrose. Somatic embryos grown in 30 g l(-1) sucrose accumulated fewer protein species than those grown in 60 g l(-1). Selected proteins were identified following mass spectrometry (nano-LC-MS/MS). Four enzymes involved in glycolysis (UDP-glucose pyrophosphorylase, fructose bisphosphate aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase GAPDH) were specifically induced in somatic embryos. 11S globulin proteins identified by MS were present in high levels in somatic embryos, zygotic embryos and endosperm, whereas 7S globulins were detected mainly in endosperm and zygotic embryos. These are the first storage proteins identified in C. persicum. Xyloglucans are known to be another group of seed storage compounds in C. persicum. Interestingly, xyloglucan endotransglycosylases were found to be highly expressed in endosperm tissue. We discuss the physiological implications of these observations.
User Published Research Carapito C, Muller D, Turlin E, Koechler S, Danchin A, Van Dorsselaer A, Leize-Wagner E, Bertin PN, Lett MC. Identification of genes and proteins involved in the pleiotropic response to arsenic stress in Caenibacter arsenoxydans, a metalloresistant beta-proteobacterium with an unsequenced genome. Biochimie. 2006 Jun;88(6):595-606.
The effect of high concentrations of arsenic has been investigated in Caenibacter arsenoxydans, a beta-proteobacterium isolated from an arsenic contaminated environment and able to oxidize arsenite to arsenate. As the genome of this bacterium has not yet been sequenced, the use of a specific proteomic approach based on nano-high performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) studies and de novo sequencing to perform cross-species protein identifications was necessary. In addition, a random mutational analysis was performed. Twenty-two proteins and 16 genes were shown to be differentially accumulated and expressed, respectively, in cells grown in the presence of arsenite. Two genes involved in arsenite oxidation and one in arsenite efflux as well as two proteins responsible for arsenate reduction were identified. Moreover, numerous genes and proteins belonging to various functional classes including information and regulation pathways, intermediary metabolism, cell envelope and cellular processes were also up- or down-regulated, which demonstrates that bacterial response to arsenic is pleiotropic.
User Published Research Fukuyama Y, Iwamoto S, Tanaka K. Rapid Sequencing and Disulfide Mapping of Peptides Containing Disulfide Bonds by Using 1,5-Diaminonaphthalene as a Reductive Matrix. J Mass Spectrom. 2006 Feb;41(2):191-201.
MS/MS is indispensable for the amino acid sequencing of peptides. However, its use is limited for peptides containing disulfide bonds. We have applied the reducing properties of 1,5-diaminonaphthalene (1,5-DAN) as a MALDI matrix to amino acid sequencing and disulfide bond mapping of human urotensin II possessing one disulfide bond, and human guanylin possessing two disulfide bonds. 1,5-DAN was used in the same manner as the usual MALDI matrices without any pre-treatment of the peptide, and MS/MS was performed using a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI QIT TOFMS). The results demonstrated that MS/MS of the molecular ions reduced by 1,5-DAN provided a series of significant b-/y-product ions. All 11 amino acid residues of urotensin II were identified using 1,5-DAN, while only 5 out of 11 residues were identified using 2,5-dihydroxybenzoic acid (DHB); similarly 11 out of 15 amino acid residues of guanylin were identified using 1,5-DAN, while only three were identified using DHB. In addition, comparison of the theoretical and measured values of the mass differences between corresponding MS/MS product ions using 1,5-DAN and DHB narrowed down the possible disulfide bond arrangement candidates. Consequently, 1,5-DAN as a reductive matrix facilitates rapid amino acid sequencing and disulfide mapping for peptides containing disulfide bonds.
User Published Research Birrell GW, Earl S, Masci PP, de Jersey J, Wallis TP, Gorman JJ, Lavin MF. Molecular diversity in venom from the Australian Brown snake, Pseudonaja textilis. Mol Cell Proteomics. 2006 Feb;5(2):379-89.
Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A2s, and pre- and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two-dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, gamma-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents.
User Published Research Siroy A, Molle V, Lemaître-Guillier C, Vallenet D, Pestel-Caron M, Cozzone AJ, Jouenne T, and Dé E. Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter Baumannii. Antimicrob Agents Chemother. 2005 Dec;49(12):4876-83.
It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (M(w) and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a beta-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels.
User Published Research Schmelzer CE, Getie M, Neubert RH. Mass Spectrometric Characterization of Human Skin Elastin Peptides Produced by Proteolytic Digestion with Pepsin and Thermitase. J Chromatogr A. 2005 Aug 12;1083(1-2):120-6.
This study investigated peptides resulting from the digestion of human skin elastin with pepsin and thermitase. Characterization of the peptides was performed using two complementary mass spectrometric techniques; LC/ESI-ion trap and nano-ESI-qTOF MS. 155 different peptides were identified using a combined database based and de novo sequencing approach resulting in a total sequence coverage of 65.4% calculated on the basis of the precursor tropoelastin (accession number A32707). A potential hydroxylation was found in 29% of the recovered prolines. Furthermore, the absence of amino acids expressed by exon 26A could be confirmed. However, contrary to earlier studies, amino acids expressed by exon 22 seem to exist.
User Published Research Getie M, Schmelzer CE, Neubert RH. Characterization of peptides resulting from digestion of human skin elastin with elastase. Proteins. 2005 Nov 15;61(3):649-57.
Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.
User Published Research Castro AJ, Carapito C, Zorn N, Magné C, Leize E, Van Dorsselaer A, Clément C. Proteomic Analysis of Grapevine (Vitis vinifera L.) Tissues Subjected to Herbicide Stress. J Exp Bot. 2005 Nov;56(421):2783-95.
Two-dimensional gel electrophoresis coupled to mass spectrometry analysis was used to examine for the first time the effect of a herbicide (flumioxazin) on a crop species (Vitis vinifera L.) at the proteome level. Examination of 2-D maps derived from chemically stressed tissues revealed the presence of 33 spots displaying a differential expression pattern. The presence of stress responsive proteins in the different plant organs analysed suggests that flumioxazin could act systemically. Among the responsive proteins, some photosynthesis-related proteins, including several fragments of the enzyme Rubisco, were identified. This effect suggests that photosynthesis could be impaired by the herbicide. The induction of several enzymatic antioxidant systems was also observed, probably as a result of an oxidative stress. Moreover, the photorespiration pathway was stimulated, as suggested by the induction of some key enzymes involved in this process. Changes in carbon metabolism-associated proteins presumably reflect altered patterns of carbon flux in response to impaired photosynthesis and an increased need for osmotic adjustment in affected tissues. Finally, plant defences were stimulated as revealed by the induction of a set of proteins belonging to the pathogenesis-related 10 class, suggesting that they could play an essential role in cell defence mechanisms against flumioxazin.
User Published Research Chen YR, Chen CL, Zhang L, Green-Church KB, Zweier JL. Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation. J Biol Chem. 2005 Nov 11;280(45):37339-48.
Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O(2)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O(2)(.) generation. O(2)(.) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O(2)(.)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200)-Ala-Gly-Ala-Tyr-Ile-Cys(206)-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)-Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.
User Poster Research Locke J, Rogalski J, Guo L, Ma B, Kast J, Lajoie G. Automated de novo Sequencing Using ToF-ToF MS/MS Data. ASMS 2005.
Peptide de novo sequencing using tandem mass spectrometry data is a standard approach in proteomics. However, most of the automated de novo sequencing software including the software provided by the mass spectrometer manufacturers, is designed for de novo sequencing using Q-ToF and ion-trap generated data. Due to the different peptide fragmentation in ToF-ToF instruments, it was uncertain whether or not the de novo sequencing software PEAKS, using parameters optimized for other instruments, will work for ToF-ToF data. In this poster we demonstrate that the Q-ToF based internal parameters of the PEAKS software work very well for both de novo sequencing and protein identification even on the average quality of ToF-ToF MS/MS data.
User Published Research Getie M, Schmelzer CE, Weiss AS, Neubert RH. Complementary mass spectrometric techniques to achieve complete sequence coverage of recombinant human tropoelastin. Rapid Commun Mass Spectrom. 2005;19(20):2989-93.
Tropoelastin is the precursor of elastin, which is a primary component of elastic fibers that provide elasticity and resilience to elastic tissues such as skin, blood vessels, lung, and ligaments. Several pathologyical conditions, such as wrinkling and sagging in sun exposed skin, Williams syndrome, supravalvular aortic stenosis, emphysema, aneurysms, and atherosclerosis, are associated with abnormalities in elastin.
User Published Research Kayser JP, Vallet JL, Cerny RL. Defining parameters for homology-tolerant database searching. J Biomol Tech. 2004 Dec;15(4):285-95.
De novo interpretation of tandem mass spectrometry (MS/MS) spectra provides sequences for searching protein databases when limited sequence information is present in the database. Our objective was to define a strategy for this type of homology-tolerant database search. Homology searches, using MS-Homology software, were conducted with 20, 10, or 5 of the most abundant peptides from 9 proteins, based either on precursor trigger intensity or on total ion current, and allowing for 50%, 30%, or 10% mismatch in the search. Protein scores were corrected by subtracting a threshold score that was calculated from random peptides. The highest (p < .01) corrected protein scores (i.e., above the threshold) were obtained by submitting 20 peptides and allowing 30% mismatch. Using these criteria, protein identification based on ion mass searching using MS/MS data (i.e., Mascot) was compared with that obtained using homology search. The highest-ranking protein was the same using Mascot, homology search using the 20 most intense peptides, or homology search using all peptides, for 63.4% of 112 spots from two-dimensional polyacrylamide gel electrophoresis gels. For these proteins, the percent coverage was greatest using Mascot compared with the use of all or just the 20 most intense peptides in a homology search (25.1%, 18.3%, and 10.6%, respectively). Finally, 35% of de novo sequences completely matched the corresponding known amino acid sequence of the matching peptide. This percentage increased when the search was limited to the 20 most intense peptides (44.0%). After identifying the protein using MS-Homology, a peptide mass search may increase the percent coverage of the protein identified.
User Published Research Blight SK, Larue RC, Mahapatra A, Longstaff DG, Chang E, Zhao G, Kang PT, Green-Church KB, Chan MK, Krzycki JA. Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo. Nature. 2004 Sep 16;431(7006):333-5.
Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.
User Poster Research Doherty-Kirby A, Booy A, Olafson B, Lajoie G, Ma B. A Comprehensive Comparison of the de novo Sequencing Accuracies of PEAKS and Other Software. ASMS 2004.
We compared three commonly used de novo sequencing programs, PEAKS, BioAnalyst and PLGS. The result showed that PEAKS has the best accuracy.