Mass Spectrometry-Based Analyses of Human Salivary Subproteomes

Saliva, an easily sampled body fluid, contains a wealth of information, coded in terms of protein composition. Cataloguing the salivary proteome of healthy individuals is the requisite first step that will allow us to link changes to specific disease states. Here, we summarize recent experiments in which we used targeted strategies to isolate several salivary subproteomes, which we analyzed using mass spectrometric (MS) approaches. Objective: Compilation of a comprehensive catalogue of proteins and peptides in human parotid (P) and submandibular/sublingual (SMSL) salivas collected as the ductal secretions from healthy donors. Methods: First, we used a variety of techniques, either separately or in combination, to fractionate the proteins in P and SMSL saliva: 2D-SDS-PAGE, multidimensional liquid chromatography (LC), metal-ion affinity separation and ultrafiltration. Then, we identified proteins and peptides in these subfractions by using a combination of MALDI-TOF-MS, MALDI-TOF/TOF-MS/MS and LC-MS/MS approaches. Next, we investigated diurnal effects on salivary composition by using a novel stable isotope dilution derivatization approach (iTRAQ) to differentially label samples collected at four time points. Results: The experiments revealed a wealth of new data, including several novel protein components, a novel phosphorylation site on the proline-rich glycoprotein and a suite of native peptides. One of the newly identified peptides has candidacidal activities. In the iTRAQ study, we identified and quantified nineteen novel peptides, many of which showed diurnal variations. Conclusions: Recent technological advances in MS-based strategies make it possible to identify the composition of proteomes and track quantitative changes as a function of important variables, such as time of day. Our growing understanding of the salivary proteome will give us the opportunity to identify and characterize changes in salivary composition that occur in various conditions, an important new strategy for gaining valuable insights into the molecular pathophysiology of oral and perhaps systemic diseases. Support: NIH-U01-DE04007; Sandler-New-Technology-Fund