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.

Figure 1

SDS-PAGE of phosphorylated proteins whole saliva. (A) Whole saliva (20 μg) was pre-treated with 1 unit of alkaline phosphatase (+AP, lane 1), heat-inactivated alkaline phosphatase (mock, lane 2), or without alkaline phosphatase (-AP, lane 3). The samples were separated by SDS-PAGE and stained with either the phospho-specific ProQ diamond dye (Left Panel) or for total protein with Sypro Ruby dye (Right Panel). Arrows indicate bands sensitive to alkaline phosphatase treatment. (B) Whole saliva was treated with Proteominer (PM) beads and incubated overnight with shaking. The unbound fraction was collected and protein bound to the beads was washed and eluted. Whole saliva (WS) starting material, the unbound fraction (Proteominer, FT), and the eluted fraction (Proteominer, EL) were separated by SDS-PAGE and stained with ProQ Diamond dye.

Figure 2

Representative tandem mass spectra of histatin 1 phosphopeptides. (A) CID spectrum identified as FYGDYGsNYLYDN, where Ser-51 is phosphorylated. (B) ETD spectrum identified as HHsHREFPFYGDY, where Ser-39 is phosphorylated. (C) CID spectrum identified as MISADsHEKR, where Ser-21 is phosphorylated. (D) CID spectrum of a synthesized form of MISADsHEKR for comparison, where Ser-21 is phosphorylated. Also displayed is the full amino acid sequence of the histatin 1 gene product with signal peptide and mature sequences marked. Residues in bold were identified by the spectra from the figure and residues in lower case are identified phosphorylation sites. Also shown at the top of the figure is the complete primary amino acid sequence of histatin 1 including with the signal peptide and mature protein sequence.

Figure 3

Representative tandem mass spectra generated by CID of phosphophoryaltion states of statherin peptides. (A) Spectrum identified as DsSEEKFLR, where Ser-21 is phosphorylated. (B) Spectrum identified as DSsEEKFLR, where Ser-22 is phosphorylated. (C) Spectrum identified as MIGADssEEKFLR, where Ser-21 and Ser-22 are phosphorylated. (D) Spectrum of a synthesized form of MIGADssEEKFLR for comparison, where Ser-21 and Ser-22 are phosphorylated. Also displayed is the full amino acid sequence of the statherin gene product with signal peptide and mature sequences marked. Residues in bold were identified by the spectra from the figure and residues in lower case are identified phosphorylation sites. The complete primary amino acid sequence of statherin including that of the signal peptide and mature protein is displayed at the top.

Figure 4

Cellular localization comparison of whole saliva proteins. Total identified proteins (black bars), phosphoproteins (open bars), and N-linked glycoproteins (gray bars) from whole saliva were organized into cellular compartments using Ingenuity Pathways Analysis^TM. The number of proteins and percentage of total are listed next to each grouping. The total salivary proteins are from . The salivary glycoproteins are from .

Figure 5

Sequence alignment comparison salivary protein phosphorylation sites. The sequence windows from 6 residues upstream and downstream surrounding the phosphorylation sites were gathered for the entire human phosphoprotein from www.phosphosite.org, (upper panel), the salivary phosphoproteome where the validity of localization was determined manually (middle panel), and the plasma phosphoproteome from (lower panel). Sequences were aligned and amino acid occurrence frequencies are represented by the size of the box containing the respective 1 letter code.