ITRAQ approach

It has been demonstrated also that the iTRAQ tandem mass spectrometric quantitative analysis strategy can be used in conjunction with the quadrupole ion trap by performing multiple stages of mass analysis (that is, MS ) [125], For example, chemical derivatization with the iTRAQ reagent not only labels the N-terminus of a peptide, but the lysine side chain also. Thus, tryptic peptides with a modified lysine residue present at the C-terminus will produce a yj product ion at m/z 291 following ClD-tandem mass spectrometry. To generate the low m/z iTRAQ reporter ions required for quantitation, the yj product ion is isolated and subjected to data-dependent CID-MS. Using this approach, peptide identification is achieved in the MS/MS scan, while quantitation is achieved via MS. ... 

Both the ICAT (Jenkins 2006) and ITRAQ methodologies (Ross 2004) can be adapted to enable absolute rather than relative quantitation of proteins, essentially by tagging a (natural isotope) synthetic version of the peptide chosen as the surrogate analyte with one of the labeled reagents. There is no question that such approaches can work well but they suffer from the same disadvantages as when used for relative quantitation and, moreover, are more laborious than some more recent methods described below. 

The membrane-bound cytochrome P450 enzymes represent key players in secondary metabolite pathways as they catalyze vital, often rate-limiting steps. However, basic, hydro-phobic and membrane-spanning proteins remain an intrinsic limitation for 2D-PAGE [118]. Adoption of gel free proteomic approaches such as iTRAQ (Table 6) may help to overcome this bottleneck. 

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