Characterization of the Human Cerebrospinal Fluid Phosphoproteome by Titanium Dioxide Affinity Chromatography and Mass Spectrometry

Abstract

Biomarkers in the cerebrospinal fluid (CSF) may be important for the diagnosis of chronic degenerative disorders in the central nervous system including dementia. Existing CSF biomarkers for dementia, however, are relatively nonspecific. More specific markers may be found by targeting investigations based on knowledge of the molecular pathology of the disease in question. In Alzheimer's disease, hyperphosphorylation of the tau protein is a characteristic feature and thus a comprehensive characterization of the phosphoproteome of the CSF may be pursued to obtain a complete picture of phosphorylation aberrations in health and disease. Toward that goal we here describe a method for a comprehensive isolation and identification of phosphorylated tryptic peptides derived from CSF proteins using a simple sample preparation step and titanium dioxide-affinity chromatography followed by MALDI-TOF or LC-MS/MS linear ion-trap-FT mass spectrometry. Whereas not all previously reported phosphoproteins were found in normal CSF, we detected 56 putative novel phosphorylation sites in 38 proteins in addition to known sites. The approach seems to be a promising foundation for the discovery of new biomarkers embedded in the CSF phosphoproteome.

Dynamic regulation of FGF23 by Fam20C phosphorylation, GalNAc-T3 glycosylation, and furin proteolysis

“…Because Fam20C localizes within the secretory pathway and the vast majority of secreted phosphoproteins are phosphorylated on S-x-E/pS motifs, Fam20C has been proposed to play a major role in the generation of the secreted phosphoproteome (5-7). For example, ∼75% of human serum and cerebrospinal fluid phosphoproteins are phosphorylated on S-x-E/pS motifs (8,9). This includes proteins important for tooth and bone formation, as well as numerous hormones.…”

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).…”

Extracellular Protein Phosphorylation, the Neglected Side of the Modification

“…Though very powerful, selectivity of these methods in CSF or serum is far below that reported for whole cell lysates. In the latter, selectivity as high as 90% could be achieved (34) whereas in body fluids selectivity is rather around 30 -60% (26,27,29), a consequence of the high dynamic range and the overall low extent of the modification. Additionally, TiO 2 and recently also IMAC was shown to isolate sialylated glycopeptides under certain conditions (35,36,29).…”

“…There is only a limited number of such reports yet and the phosphorylation data derived from these studies are far from those targeting intracellular phosphorylation. In cerebrospinal fluid (CSF) 25-45 phosphoproteins were identified from 60 -200 g starting material combining SCX fractionation with phosphoSer specific molecularly imprinted polymers or using TiO 2 enrichment (25,26). Plasma/serum phosphoproteome analyses fared even worse in this aspect, 40 -100 l of plasma (ϳ2-5 mg protein) yielded 30 -70 phosphoproteins using TiO 2 enrichment (either alone or in combination with SCX) or Fe(III)-IMAC (27)(28)(29).…”

“…However, comparison of TiO 2 enrichment applied to serum with or without depletion of albumin and IgG showed considerable loss of phosphopeptides in the depleted fraction (27). Depletion of the highly abundant proteins was also found detrimental in the phosphoproteome analysis of CSF (26). Indeed, off-target removal was reported using albumin-specific and other immunodepletion kits (31)(32)(33).…”