Stable isobaric labelling is the best way to enable accurate simultaneous analysis of multiple samples by mass spectrometry (MS) based proteomics. Currently the method which allows the most multiplexicity, isobaric labelling exists as two variants, Tandem Mass Tag (TMT) and Isobaric tags for relative and absolute quantitation (iTRAQ).
In isobaric labelling, peptides obtained following digestion of the protein samples and are labelled with small isotopically-labelled molecule tags.
After the different samples are combined, the tags are indistinguishable until they are specifically fragmented and quantified by MS. This allows greater sensitivity than with SILAC, where the different channels are perceived as different peaks by the MS from the start.
By allowing simultaneous MS analysis of up to 11 different samples, isobaric labelling allows highly accurate relative quantitation as well as significant reductions in costs and instrument time.
Isobaric labels are usually amine-reactive (iodo-TMT is also available for cysteine-reactivity) so will label non-acetylated peptide N-termini and Lysine residues.
Although labelling is normally performed at the peptide level, it can also be performed pre-digestion at the protein level, however this will block tryptic cleavage after Lysine residues.
NB: Isobaric labelling works best with mass spectrometers which allow MS3-level quantitation (e.g. Thermo’s Fusion Orbitraps). The additional filtering step allows almost complete correction of so called “ratio compression”, an issue which arises due to contamination of MS1 precursors by other peptides with different ratios.