In this technique, the introduction of stable isotopes using iTRAQ reagents occurs on the level of proteolytic peptides ( Figure 3 ). This technology uses an NHS ester derivative to modify primary amino groups by linking a mass balance group (carbonyl group) and a reporter group (based on N-methylpiperazine) to proteolytic peptides via the formation of an amide bond [117] . Due to the isobaric mass design of the iTRAQ reagents, differentially labelled peptides appear as a single peak in MS scans, reducing the probability of peak overlapping. When iTRAQ-tagged peptides are subjected to MS/MS analysis, the mass balancing carbonyl moiety is released as a neutral fragment, liberating the isotope-encoded reporter ions which provides relative quantitative information on proteins. An inherent drawback of the reported iTRAQ technology is due to the enzymatic digestion of proteins prior to labelling, which artificially increases sample complexity and this approach needs a powerful multidimensional fractionation method of peptides before MS identification.