Phosphoproteome Analysis of Human Liver Tissue by Long-gradient Nanoflow LC Coupled with Multiple Stage Mass Spectrometric Analysis

Reversible protein phosphorylation plays a critical role in liver development and function. Comprehensively cataloging the phosphoproteins and their phosphorylation sites in human liver tissue will facilitate the understanding of physiological and pathological mechanisms of liver. As lacking of efficient approach to fractionate phosphopeptides, nanoflow-RPLC with long gradient elution was applied to reduce the complexity of the phosphopeptides in this study. Two approaches were performed to further improve the coverage of phosphoproteome analysis of human liver tissue. In the first approach, 10 replicated long-gradient LC-MS/MS runs were performed to analyze the enriched phosphopeptides which resulted in the localization of 1,080 phosphorylation sites from 495 proteins. In the second approach, proteins from liver tissue were first fractionated by SDS-PAGE and then long-gradient LC-MS/MS analysis was performed to analyze the phosphopeptides derived from each fraction which resulted in localization of 1,786 phosphorylation sites from 911 proteins. The two approaches showed the complementarity in phosphoproteome analysis of human liver tissue. Combining the results, the identification of 2,225 nonredundant phosphorylation sites from 1,023 proteins was obtained. The confidence of phosphopeptide identifications was strictly controlled with false discovery rate (FDR) ≤1% by a MS2/MS3 target-decoy database search approach. Among the localized 2,225 phosphorylated sites, as many as 70.07% (1,559 phosphorylated sites) were also reported by others which confirmed the high confidence of the sites determined in this study. Considering the data acquired from low accuracy mass spectrometer and processed by a conservative MS2/MS3 target-decoy approach, the high confidence phosphoproteome data set obtained for human liver tissue in this study is quite impressive