Relative Quantitative Phosphoproteome Of Whole Saliva Supernatant

RELATIVE QUANTITATIVE PHOSPHOPROTEOME OF WHOLE SALIVA SUPERNATANT BY MASS SPECTROMETRY.

H.NOUH, F.G.OPPENHEIM and E.SALIH, BOSTON UNIVERSITY, SCHOOL OF DENTAL MEDICINE, MA, USA

Phosphoproteins with significant biological functions have been identified and characterized in whole saliva supernatant (WSS). Quantitative analysis permits comparison between healthy individuals and those with periodontal disease and this can be done on a large scale using state-of-the-art mass spectrometric technology. Objective: Quantitative large-scale the phosphoproteome of WSS using high throughput mass spectrometric techniques and validation with a luminex istrument. Methods: Whole saliva collection was carried out under masticatory stimulation using  1.5 g of parafilm and after collection the samples were centrifuged at 14,000 x g for 20 min to separate the supernatant from the sediment. We utilized  dithiothreitol (DTT) and its deutarated isotopic form ([D₆] DTT for chemical derivatization of the phosho-serine/threonine containing proteins and covalent  chromatography for phosphopeptides enrichment. WSS samples from 40 healthy subjects and 40 periodontal patients were used to generate 4 pools each derived from 10 healthy controls and 10 periodontal patients. Equal amounts of protein of healthy and periodontal samples were trypsin digested seperatley and derivatized by DTT (healthy) and DTT [D₆] (periodontal samples). The healthy and periodontal samples were combined and the phosphopeptides were enriched by covalent-chromatography followed by nano-flow LC-ESI MS/MS analysis. A selected group of  proteins were validated using a multiplex approach. Results: Our work led to the identification and quantification of over 50 phosphoproteins in WSS. The used approaches revealed the identity of the phosphoprotein and the precise site(s) of phosphorylation. It also permitted relative quantitative levels of each phosphoprotein in both samples. Conclusion: Validation experiments confirmed that the MS data and the results obtained have important implications for salivary functions and the discovery of useful biomarkers for oral and systemic diseases. Supported by NIH/NIDCR Grants DE018448; DE05672; DE07652.