An Initial Characterization of the Serum Phosphoproteome

Abstract

Phosphorylation is a dynamic post-translational protein modification that is the basis of a general mechanism for maintaining and regulating protein structure and function, and of course underpins key cellular processes through signal transduction. In the last several years, many studies of large-scale profiling of phosphoproteins and mapping phosphorylation sites from cultured human cells or tissues by mass spectrometry technique have been published; however, there is little information on general (or global) phosphoproteomic characterization and description of the content of phosphoprotein analytes within the circulation. Circulating phosphoproteins and phosphopeptides could represent important disease biomarkers because of their well-known importance in cellular function, and these analytes frequently are mutated and activated in human diseases such as cancer. Here we report an initial attempt to characterize the phosphoprotein content of serum. To accomplish this, we developed a method in which phosphopeptides are enriched from digested serum proteins and analyzed by LC-MS/MS using LTQ-Orbitrap (CID) and LTQ-ETD mass spectrometers. Using this approach we identified ~100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate.

Crystal structure of the Golgi casein kinase

“…One member of this family, Fam20C (family with sequence similarity 20, member C), is the physiological casein kinase that phosphorylates multiple secreted proteins within a Ser-x-Glu/pSer motif (7,9,10). The importance of this discovery is underscored by the fact that some 75% of the phosphoproteins identified in human serum and cerebrospinal fluid contain phosphate within this motif (11)(12)(13). Furthermore, mutations in FAM20C cause Raine syndrome, a deadly osteosclerotic bone dysplasia characterized by generalized osteosclerosis, ectopic calcifications, and characteristic facial features (14)(15)(16).…”

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.…”

“…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).…”

“…Recently, human serum phosphoproteome has already been explored by several labs. Zhou et al enriched phosphopeptides from tryptic digest of serum sample by TiO 2 and identified about 100 unique phosphopeptides after LC-MS/MS analysis [9]. Meanwhile, Carrascal et al applied strong cation exchange (SCX) for fractionation of phosphopeptides enriched from serum protein digest.…”