What is De Novo Sequencing?

De novo sequencing, peptide sequences are derived from the masses of their fragments as shown on a tandem mass spectrum. When performing de novo sequencing, no protein sequence database is used for reference.¹

Click here for a A Quick Introduction to Peptide De Novo Sequencing.

Discovery and Confirmation

PEAKS is renowned for its robust de novo sequencing module. De novo sequencing is becoming highly regarded as a standard method of validation utilized in the identification process of known sequences, due to its ability to provide confidence to database results. The PEAKS method sequences each spectrum through de novo sequencing, followed by performing a PEAKS database search. The results from de novo sequencing are automatically considered by the database algorithm, aiding in the removal of false positives. De novo sequencing is not just for discovery.

For studying unsequenced organisms, de novo sequencing, manually or automated, is the only method for identification, as no database will contain these sequences. PEAKS provides users the ability to perform automated de novo sequencing, as well as a manually-assisted approach to de novo sequencing to accurately identify sequences of known and unknown species.

If your lab is not using de novo sequencing on a regular basis, be sure to read the list of limitations of single database dependency.

• Good quality spectra are often left unexplained after database searching; de novo sequencing is the only recourse.
• Since a database search cannot reliably find peptides that are unexpectedly modified, de novo sequence information is a valuable first step in finding these PTM.
• A de novo sequencing based approach is proven to be the best method database search results validation. False positives are virtually eliminated.
• Sequence tag searching requires good quality de novo sequences to be effective. Coverage can be drastically increased by using a sequence tag based and MS/MS ion searches together.
• An incomplete or corrupted database is a common, but invisible problem in protein identification and characterization research. How many peptides are you missing because of this? De novo sequencing as a regular part of your workflow will help catch these.
• When studying species that are not in the public databases, de novo sequencing is the only way to get valuable peptide sequence information.

The core to PEAKS technology is the fastest, most accurate auto de novo algorithm available on the market. Run auto de novo on an entire LC run, or a batch of them - process data faster than you can make it (40+ spectra per second). The results are unmatched in accuracy; PEAKS determines at least 3 times as many completely correct sequences as the next best de novo software. Accurate mass capabilities mean de novo at 97% accuracy is possible.

PEAKS reports reliable and straightforward confidence scores. Some unique features of PEAKS de novo are confidence scores at the amino acid level. PEAKS provides an overall score for each and every de novo tag generated, every amino acid contained within that tag is given a confidence score so you can clearly gauge the quality of each and every hit. PEAKS can work with a, b, c and x, y, z product ions and this is something only very few programs can boast. PEAKS has a wide range of built in enzyme options, including a no‐enzyme digest option. Not only that, but PEAKS also allows users to define their own enzymes with custom cleavage rules. PEAKS de novo can also consider PTMs in its analysis within both the automated and manual de novo sequencing methods.

The objective of the PEAKS software is to provide users with the greatest control over their data, while providing in depth analysis, without cluttering up the display.

PEAKS is the only de novo sequencing software specifically tuned to handle data from QTOF, TOF-TOF, Ion Trap, Orbitrap and FTMS instruments, while using y/b ions or c/z ions for fragmentation flexibility. It provides a reliable central processing tool for all instrument and experiment types.

As you will quickly realize, PEAKS provides much more than de novo sequencing. In addition to de novo sequencing, PEAKS has an integrated database search engine for protein identification (PEAKS Search), multi-search engine protein ID (inChorus), homology searching (SPIDER), Quantification (PEAKS Q), PTM identification (PTM Finder) and much more.

FOOTNOTES:

1. Nature. Glossary, 2010.