Quantitative serum proteomics using dual stable isotope coding and nano LC-MS/MSMS

Stable isotope coding technique in combination with mass spectrometry has emerged as a powerful tool to accurately identify and differentially quantify proteins within complex protein mixtures. We present a novel methodology to increase the yield of quantified proteins while maintaining a high stable-isotopic labeling efficacy. With this approach, intact proteins in complex biological sample such as sera are labeled with the designated dual stable isotope coding (DSIC) systems. In brief, intact proteins are coded sequentially with acrylamide to label Cysteine residues (Cys) and with succinic anhydride to label Lysine residues (Lys). Protein samples coded with this dual stable isotope are subjected to an online 2D-HPLC fractionation. The resolved protein fractions are individually digested with trypsin and analyzed with nano LC-MS/MSMS. Our results show that the DSIC labeling efficiency is 100% for Cysteine (Cys) labeled with acrylamide and 98% for Lysine (Lys) labeled with succinic anhydride. A comparative analysis of DSIC labeling and single labeling of Cysteine residues was made. Analysis of an entire anion-exchange chromatography subfraction of sera yielded 165 identified proteins (criteria: error rate <5% and unique peptides ≥2), 104 of which were quantified using the single labeling method (i.e., Cysteine acrylamide labeling only). In contrast, using same criteria for identification, a total 185 proteins were identified and 174 proteins were quantified using the DSIC labeling technique.