Robust workflow for iTRAQ-based peptide and protein quantification.

Quantitative proteomics has become a routinely used technique to globally compare protein content and expression profiles of biological samples, for instance after differential stimulation. In this context, chemical stable isotope-based labeling techniques, such as ICAT and iTRAQ, have been successfully applied in a large variety of studies. Since iTRAQ labels are isobaric, quantitation is conducted on the MS/MS level. Consequently, up to eight samples can be multiplexed and quantified in a single experiment without increasing sample complexity. Here, we present a robust workflow to conduct iTRAQ quantification of biological samples such as human platelets, which comprises (a) an adequate sample preparation procedure, (b) an optimized tryptic digestion protocol, (c) SPE desalting and subsequent peptide labeling using a 4-plex iTRAQ labeling kit, and (d) fractionation of the obtained peptide mixture by strong cation exchange chromatography.