ICAT Quantification

Isotope-coded affinity tags (ICATs) are a gel-free method for quantitative proteomics that relies on chemical labeling reagents.

These chemical probes consist of three general elements: a reactive group capable of labeling a defined amino acid side chain (e.g., iodoacetamide to modify cysteine residues), an isotopically coded linker, and a tag (e.g., biotin) for the affinity isolation of labeled proteins/peptides. For the quantitative comparison of two proteomes, one sample is labeled with the isotopically light (d0) probe and the other with the isotopically heavy (d8) version. To minimize error, both samples are then combined, digested with a protease (i.e., trypsin), and subjected to avidin affinity chromatography to isolate peptides labeled with isotope-coded tagging reagents. These peptides are then analyzed by liquid chromatography-mass spectrometry (LC-MS). The ratios of signal intensities of differentially mass-tagged peptide pairs are quantified to determine the relative levels of proteins in the two samples.

SILAC Quantification

Stable isotope labeling with amino acids in cell culture (SILAC) is a method which metabolically label proteins for relative quantification comparison. One cell population is fed amino acids of normal isotopic composition; the other cell population is fed amino acids labeled with heavier isotopes. The heavy amino acids are incorporated into newly synthesized proteins, eventually completely replacing the cells' proteins, such that labeling efficiency is near 100%. The cell populations are then mixed together and digested for MS analysis to determine differential protein abundances.

iTRAQ Quantification

Isobaric tagging for relative and absolute quantification (iTRAQ) uses isotopic labeling to enable relative quantification comparisons. Up to eight different proteomic samples can be labeled using eight different isobaric tags.

User Defined Labeling Quantification

In addition to PEAKS compiling a single resource (PEAKS Q) with a variety of quantitative proteomic solutions, many users employ other suitable methods for labeling and identifying particular isotopic peaks; PEAKS Q supports these unique labeling methods.