iTRAQ is one of the newest techniques used for the quantitative study of gene expression at the proteome level. It retains post-translational modification (PTM) information, and allows multiplex analysis of up to four samples in a single run. It is ideal for (1) protein expression analysis or quantification experiments, (2) PTM analysis and (3) discovery/validation analyses for biomarker elucidation, drug target screening and time course studies.

The iTRAQ technology involves chemically tagging the N-terminus of enzymatically digested peptides that have been isolated from cells e.g. disease vs. non-disease states. The two labelled samples are then combined, separated on a LC system and analyzed by ESI-MS directly or by MALDI-TOF. A database search is then performed using the fragmentation data generated by the peptides to identify the labelled peptides and hence the corresponding proteins (itraq fig1). During the fragmentation event, the tag attached to the peptides generates a low Mwt reporter ion that is unique to the tag used to label each of the digests (iTraq Fig2). Measurement of the intensity of these reporter ions, allows the relative quantification of the peptides in each digest to be determined and consequently the proteins from which they originated. Currently there are four tags commercially available enabling four different conditions to be multiplexed together in one experiment (itraq Fig 3 ).