Proteins iTRAQ

Depletion of these and several other abundant proteins allow for in-depth analysis of low-abundance ones. There are many commercial products for protein immunodepletion. Applications of the products are reported for a Seppro column in a label-free method (135), Seppro column in a SILAP method (136), a Hu-6 Multiple Affinity Removal System (MARS) kit in an iTRAQ method (137), and a Hu-6 MARS kit in another iTRAQ -based method (138). Caution should be observed if a low-abundance protein has affinity to a high-abundance protein because it can be co-depleted. 

The technique of isobaric tag for relative and absolute quantification (iTRAQ) is based on covalently labeling the amines at N-termini and the side chains of peptides with isotope-coded tags after the proteins have been isolated form biological systems, denatured, and enzymatically digested. Here the isotope labeling is carried out at a much later stage than in both SILAC and ICAT. iTRAQ is an MS/MS technique that uses CID-derived ions for both the identification and quantification of proteins. 

Quantification and protein identification Figure 8.9. iTRAQ ICAT quantification protocol. 

MassTag (Lys) [71] and iTRAQ (Lys) [72], aU of which are suitable for MALDI-MS and MS/MS. A simple method, namely trypsin-catalyzed incorporation of 0 into carboxyl groups, fadhtates the comparative analysis of protein and peptide samples by MALDI-MS [73, 74]. 

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