PNNL

Abstract

Quantitative proteomic measurements are of significant interest in studies aimed at discovering disease biomarkers and providing new insights into biological pathways. A quantitative cysteinyl-peptide enrichment technology (QCET) can be employed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomic studies that utilize stable-isotope labeling techniques combined with high-resolution liquid chromatography (LC)-mass spectrometry (MS) measurements. The QCET approach involves specific 16O/18O labeling of tryptic peptides, high-efficiency enrichment of cysteinyl-peptides, and confident protein identification and quantification from high resolution LC-Fourier transform ion cyclotron resonance mass spectrometry (FTICR) measurements and a previously established database of accurate mass and elution time information. This methodology is demonstrated by using proteome profiling of naïve and in vitro-differentiated human mammary epithelial cells (HMEC) as an example, which initially resulted in the identification and quantification of 603 proteins in a single LC-FTICR analysis. QCET provides not only highly efficient enrichment of cysteinyl-peptides for more extensive proteome coverage and improved labeling efficiency for better quantitative measurements, but more importantly, a high-throughput strategy suitable for quantitative proteome analysis where extensive or parallel proteomic measurements are required, such as in time course studies of specific pathways and clinical sample analyses for biomarker discovery.