Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen

Reversible phosphorylation of proteins is the most common PTM in cell-signaling pathways. Despite this, high-throughput methods for the systematic detection, identification, and quanti-fication of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO₂)-based 3-D LC (strong cationic exchange/TiO₂/C18)-MS³-linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low-abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (H₂ ¹⁸O for tissues and plasma). Two examples of potential HCC phospho-biomarkers including plectin-1(phopho-Ser-4253) and alpha-HS- glycoprotein (phospho-Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO₂-based online-3-D LC-MS³-linear ion trap system with highthroughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers.