Relative Protein Quantification Using Tandem Mass Tag Mass Spectrometry

Measuring protein changes over time or following stimuli is one of the important tasks of proteomics. In the past decade, several strategies have been developed for the relative quantification of proteins using mass spectrometry (MS). Isobaric labeling strategies for relative quantitative proteomics allow for parallel multiplexing of quantitative experiments. With this technique, multiple peptide samples are chemically labeled with isobaric chemical tag variants and each variant has the same molecular structure and mass. Each variant, however, is designed to produce a unique "reporter ion" when fragmented inside a mass spectrometer. Once peptide samples are labeled, combined, and analyzed using MS, differentially labeled peptides are indistinguishable in a first, MS spectrum of intact peptides. However, since each tag variant contains a labile component with different mass, "reporter ions" can be generated and recorded in a subsequent MS2 spectrum. Intensities from each variant are recorded to represent the relative abundances of the peptide in each sample. Isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) are commercially available reagents for performing this technique. Here, we describe the general workflow of relative quantification of proteins using TMT by MS2, or an additional MS3 spectrum.