A new versatile peptide-based size exclusion chromatography platform for global profiling and quantitation of candidate biomarkers in hepatocellular carcinoma specimens

Disease biomarkers are predicted to be in low abundance; thus, the most crucial step of biomarker discovery is the efficient fractionation of clinical samples into protein sets that define disease stages and/or predict disease development. For this purpose, we developed a new platform that uses peptide‐based size exclusion chromatography (pep‐SEC) to quantify disease biomarker candidates. This new platform has many advantages over previously described biomarker profiling platforms, including short run time, high resolution, and good reproducibility, which make it suitable for large‐scale analysis. We combined this platform with isotope labeling and label‐free methods to identify and quantitate differentially expressed proteins in hepatocellular carcinoma (HCC) tissues. When we combined pep‐SEC with a gas phase fractionation method, which broadens precursor ion selection, the protein coverage was significantly increased, which is critical for the global profiling of HCC specimens. Furthermore, pep‐SEC‐LC‐MS/MS analysis enhanced the detection of low‐abundance proteins (e.g. insulin receptor substrate 2 and carboxylesterase 1) and glycopeptides in HCC plasma. Thus, our pep‐SEC platform is an efficient and versatile pre‐fractionation system for the large‐scale profiling and quantitation of candidate biomarkers in complex disease proteomes.