Ricerc@Sapienza

Protein post-translational modifications (PTMs) play important roles in biological systems and help shed light on important physiological and pathological processes. Still, most PTMs remain underinvestigated. Protein O-sulfation (sometimes also referred to as O-sulfonation) is such a PTM (tyrosine-sulfation is the most studied in the literature, but serine- and threonine O-sulfation have also been reported to exist), due to lack of analytical methods. Two are the main challenges associated with high-throughput shotgun sulfoproteomics: sample preparation and sulfopeptide identification by mass spectrometry. Detection and identification of sulfation by mass spectrometry, taking into consideration both sulfation site localization and selectivity to distinguish it from the nearly isobaric phosphorylation, is the second main challenge in sulfoproteomics.
The main goal of this project proposal is to provide a large scale workflow for identification of sulfoproteins by shotgun proteomics. It is organized into two sub-goals: the first one is to extend the analytical method developed on sulfopeptide standards to a complex real-world sample and optimize it for recovery of unknown sulfopeptides first obtained by tryptic digestion of a standard sulfoprotein (fibrinogen), then to serum and cell protein digests. The whole analytical workflow, comprising sulfopeptide enrichment and nanoHPLC MS/MS with high resolution, would fill a gap which currently exists for the characterization of this neglected PTM, for which no large scale analytical method exists for complex proteomes, despite the potential interests for the biological activity.