Characterization of the Human Plasma Phosphoproteome Using Linear Ion Trap Mass Spectrometry and Multiple Search Engines

Abstract

Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

Analysis of human serum phosphopeptidome by a focused database searching strategy

“…For example, the four most abundant phosphopeptides derived from fibrinogen alpha (T.ADpSGEGDFLAEGGGVR.G, T.ADpSG EGDFLAEGGGV.R, A.DpSGEGDFLAEGGGVR.G, A.DpSGEGDFLA EGGGV.R) are not fully tryptic peptides. They were identified only in this study but not in other proteomic studies [9,10]. CID (MS2 or MSA) is the popular fragmentation method for identifications of phosphopeptides in phosphoproteomic analysis.…”

“…Most of the endogenous phosphopeptides identified in this study are not reported in previous proteomic studies using trypsin as digestion enzyme. This is because most of these identified phosphopeptides are partial tryptic or non-tryptic peptides generated by proteases presented in serum [9,10]. For example, the four most abundant phosphopeptides derived from fibrinogen alpha (T.ADpSGEGDFLAEGGGVR.G, T.ADpSG EGDFLAEGGGV.R, A.DpSGEGDFLAEGGGVR.G, A.DpSGEGDFLA EGGGV.R) are not fully tryptic peptides.…”

“…Most of these pathways play an important role in serum. Among them, the coagulation system has been described in human plasma phosphoproteome study by Carrascal et al [10]. They found that 12 of the phosphoproteins they identified were related to the complement and coagulation cascade.…”

“…Among the 133 identified phosphorylation sites, only 20 were included in the Phosphosite Database (www.phosphosite.org). Comparing with the phosphorylation sites identified in serum proteins in literatures [9,10], only 14 were reported in 12 phosphoproteins (alpha-2-HS-glycoprotein, apolipoprotein A-IV, apolipoprotein E, apolipoprotein-L1, fibronectin, ITI heavy chain H4, matrix extracellular phosphoglycoprotein, osteopontin, selenoprotein P, SERPINC1 protein and kininogen 1). Altogether only 24 phosphorylation sites were reported by previous studies (Supporting Information 04).…”

“…After LC-MS/MS analysis, multiple search engines were applied to search the MS data. They identified 138 unique phosphopeptides, among which, 85 were identified by more than one search engine [10]. Garbis et al combined size exclusion fractionation, hydrophilic interaction chromatography and ultraperformance RPLC-MS/MS to analyze the serum proteome and phosphoproteome of patients with benign prostate hyperplasia [11].…”