Fig. 4.

Isobaric chemical labeling method. Isobaric chemical labeling, including isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tag (TMT), labeled the N-termini and the lysine-side chains in the digested peptides with different isobaric compounds, which have the same mass and chemical structure but contain different numbers and combinations of ¹³C and ¹⁵N isotopes in the mass reporter. Then, the different tags were identified and the relative peptide abundances estimated. Because the masses of all of the tags are the same, identical peptides from different samples are co-eluted and selected by MS. After tag cleavage and another round of MS, the tags are used to quantitate relative peptide intensities, while the peptide fragment ions are sequenced for protein identification. The isobaric chemical labeling based multiplexing comparison is used to compare up to four, six, eight, or ten samples, depending the isobaric tags used (i.e., 4-plex iTRAQ, 6-plex TMT, 8-plex iTRAQ, or 10-plex TMT)