Pre-Fractionation Strategies Enable Expansion of the Human Salivary Protein Catalogue

A thorough understanding of salivary composition sets the stage for biomarker discovery and improved replacement therapies. To maximize the number of protein and peptide species identified in this complex bodyfluid, enrichment steps are required to identify low-abundance components that would otherwise be obscured. To this end, saliva samples [parotid (PS) and submandibular/sublingual (SMSL)] were pre-fractionated before analysis using a shotgun-type proteomics approach. Objectives: The goal of this study was to use multidimensional liquid chromatography (LC) in conjunction with mass spectrometry (MS) in order to delve deeper into the human salivary proteome. Methods: Samples of PS and SMSL salivas were sequentially separated into three fractions: native peptides; native peptides released from higher-molecular-weight (HMW) complexes; and trypsin digestion of the HMW proteins. Then, each fraction was subjected to strong cation exchange chromatography in either a column or a spin format. Finally, the subfractions were separated by reversed phase liquid chromatography and analyzed by tandem MS/MS using either electrospray- or matrix-assisted laser desorption ionization. The MASCOT search engine was used to match MS/MS fragment ion data to individual peptide sequences in the Swissprot protein database. Results: The separation strategy described above resulted in a 4-fold increase in the number of unique precursor ions selected for MS/MS fragmentation. The number of peptide sequences identified in the LC-MS/MS analysis of PS and SMSL salivas also increased. Protein identities were dominated by known salivary components (cystatins, proline-rich proteins, and amylase) but a bevy of unique peptide masses was uncovered. Conclusions: Salivary protein components were readily identified by a shotgun-type proteomic analysis of PS and SMSL salivas. The application of pre-fractionation steps dramatically increased the number of unique peptide masses detected. We suspect that the novel components of the salivary peptidome that we have discovered play a critical role in oral health. Support: NIH-T32-DE07306; U01-DE04007; Sandler-New-Technology-Fund