Stable isotope labelling with amino acids in cell culture (SILAC) and 15N-labelling involve feeding either unlabelled, or heavy-isotope (e.g. 13C, 15N, 18O and 2 H)-enriched, amino acids to different experimental groups of cells/animals. Such labelled peptides can be distinguished by mass spectrometry, enabling relative quantitation of the ‘heavy’ and ‘light’ forms of the same peptide. Quantitative information is derived from the intact peptide ions in the MS1 spectra and peptide identities derived from MS2 spectra of fragmented peptides.

 

Isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tagging (TMT) involves conjugating separate peptide samples with a chemical tag that incorporates different combinations of isotopic labels (generating distinguishable mass signals), which are then mixed and analysed together by LC-MS/MS. Each tag has the same mass and therefore the same peptide labelled from different peptide samples will also have the same intact mass, and will appear as one ion in the MS1 spectrum. After peptide fragmentation, however, a ‘reporter ion’ from each tag is liberated to generate a set of reporter ions detected in the MS2 spectrum, each with a different mass, to be used for quantitation. Current state-of-the-art methods for reporter ion quantitation use an extra round of fragment ion isolation and fragmentation to purify the reporter ion signal (MS3 spectrum).