Stable, isobaric labelling of peptides is an elegant method to enable the simultaneous analysis and relative quantitation of multiple samples by mass spectrometry (MS)-based proteomics. Currently, the method that enables the greatest degree of multiplexing within a single experiment is the tandem mass tag (TMT).
Peptides generated by digestion of proteins are modified with small, stable, isotopically-labelled molecular tags.
After individual samples have been labelled with the different tags, the samples are combined and analysed by mass spectrometry. At this point, the mass of the same (labelled) peptide across the different samples is indistinguishable. Only after selection and fragmentation of the peptide (and release of the isobaric label) can the origin of the peptide be determined. In addition, the release of the label enables the relative quantitation of the peptide between conditions.
The TMT isobaric labels are amine-reactive and modify the N-terminus of peptides and the side chain of lysine residues. Currently, TMT reagents are available as 6-, 10-, 11- and 16-plex kits.
Relative quantitation of peptides by isobaric labels can be further refined via analysis on mass spectrometers that allow quantitation of the TMT tag at the MS3 level (e.g., ThermoFisher Scientific Orbitrap Fusion Tribrid and Orbitrap Fusion Lumos Tribrid mass spectrometers). The additional selection and fragmentation step enables almost complete elimination of what is termed “ratio compression”. This effect skews the quantitation values and arises when contaminating precursor ions from co-eluting peptides with different TMT label ratios are selected at the MS1 level.