Quantitative Serum Proteomics Using Dual Stable Isotope Coding and nano LC-MS/MSMS Hong Wang,* Chee-Hong Wong, Alice Chin, Jacob Kennedy, Qing Zhang, and Samir Hanash Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Received February 17, 2009
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
