Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis

The combination of isotope coded affinity tag (ICAT) reagents and tandem ma ss spectrometry constitutes a new method for quantitative proteomics. It in volves the site-specific, covalent labeling of proteins with isotopically n ormal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the l abeled peptides. The method critically depends on labeling protocols that a re specific, quantitative, general, robust, and reproducible. Here we descr ibe the systematic evaluation of important parameters of the labeling proto col and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonst rate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures s uch as a yeast cell lysate. (C) 2001 Academic Press.