title: Large-scale phosphoproteomics analysis of whole saliva reveals a distinct phosphorylation pattern.
abstract: In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in "extracellular" and "plasma membrane" categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.
authors.affiliation: Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Stone
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Chen
authors.forename: Thomas
authors.initials: T
authors.lastname: McGowan
authors.forename: Sricharan
authors.initials: S
authors.lastname: Bandhakavi
authors.forename: Bin
authors.initials: B
authors.lastname: Cheng
authors.forename: Nelson L
authors.initials: NL
authors.lastname: Rhodus
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Griffin
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D030562:Databases, Protein; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis* / Q000235:genetics; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012463:Saliva / Q000737:chemistry*; D012471:Salivary Proteins and Peptides / Q000032:analysis* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D010750:Phosphoproteins; D012471:Salivary Proteins and Peptides