Quantification iTRAQ

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. 

Figure 6 Principle of iTRAQ quantification. Reproduced from C. Dass, Fundamentals of Contemporary Mass Spectrometry, Wiley-lnterscience Hoboken, NJ, 2007, with permission from Wiley-lnterscience, Copyright 2007.

FIGURE 3.40 Quantification using 4-plex iTRAQ (an in vitro labeling method). 

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

Any chemistry that does not yield full incorporation of the label would obviously not be useful, but it is equally important that the reaction produce a minimum of side products. Even when the reaction conditions are closely monitored, side products can sometimes not be avoided (e.g., partial labeling of tyrosines when using the iTRAQ reagent). The side products do not necessarily cause major problems because the quantification can be... 

A few nonstandard chemical labels that have been used successfully include dimethylation of free amines (i.e., lysines and N-termini) using reductive amination, guanidination of lysines, and alkylation of cysteines by acrylamide. In practice, the commercially available labels (e.g., ICAT and iTRAQ) have received the most widespread use. Ones that are based on chemical reactions require a deeper understanding of chemistry (in contrast to the kits) and also computational skills, because there are very few generally available programs that can be used for automated quantification of the data sets. 

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