Phosphoproteome of Oral Fluids by Mass Spectrometry

Thus far only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids and these represent the abundant protein constituents of saliva. These phosphoproteins have been well characterized structurally and their important biological functions in the oral cavity have been well documented. Objective: To evaluate and determine on a large-scale phosphoproteome of oral fluids using high-throughput mass spectrometric approaches. Method: We have utilized a readily available bi-functional thiol-reagent, dithiothreitol (DTT), for chemical derivatization of the phospho-serine/threonine containing proteins and covalent disulphide-thiol-interchange chromatography to capture and enrich phosphopeptides. Whole saliva (WS) samples from five healthy individuals were used for tryptic digestion, chemical derivatization by DTT, and enrichment of phosphopeptides by disulphide-thiol-interchange covalent-chromatography followed by nano-flow LC-ESI-MS/MS analysis. Parotid saliva from four individuals were treated similarly. Results: The combined chemical strategy and state-of-the-art mass spectrometry led to the identification of 116 phosphoproteins in WS most of which were hitherto unknown phosphoprotein components. Parotid saliva on the other hand contained only about one third of the phosphoproteins found in WS. The specific approaches used in this study permitted sequence identity of the specific phosphopeptide region(s) and the precise site(s) of phosphorylation. Conclusion: This study is the first to move the thiol-based chemistries beyond the stage of “proof-of-principle” for global phosphoproteomics using mass spectrometry. The identified phosphoproteins also represent the first documentation of a large-scale phosphoproteome of WS and parotid saliva. These unique phosphoproteins cover a wide range of biological and molecular functions that are likely to contribute significantly to the overall biology of the oral cavity. The results obtained are not only important for salivary function but also for the discovery of biomarkers useful for diagnostic purposes. Supported by NIH/NIDCR Grants DE05672; DE07652; DE14950.