Abstract
Some glycoproteins contain carbohydrates S-linked to cysteine (Cys) residues. However, relatively few S-glycosylated proteins have been detected, due to the lack of an effective research methodology. This work outlines a general concept for the detection of S-glycosylation sites in proteins. The approach was verified by exploratory experiments on a model mixture of β-S-glucosylated polypeptides obtained by the chemical transformation of lysozyme P00698. The model underwent two processes: (1) oxidative hydrolysis of S-glycosidic bonds under alkaline conditions to expose the thiol group of Cys residues; (2) thiol S-alkylation leading to thiol S-adduct formation at the former S-glycosylation sites. Oxidative hydrolysis was conducted in aqueous urea, dimethyl sulfoxide, or trifluoroethanol, with silver nitrate as the reaction promoter, in the presence of triethylamine and/or pyridine. The concurrent formation of stable protein silver thiolates, gluconic acid, and silver nanoclusters was observed. The essential de-metalation of protein silver thiolates using dithiothreitol preceded the S-labeling of Cys residues with 4-vinyl pyridine or a fluorescent reagent. The S-labeled model was sequenced by tandem mass spectrometry to obtain data on the modifications and their distribution over the protein chains. This enabled the efficiency of both S-glycosidic bonds hydrolysis and S-glycosylation site labeling to be evaluated. Suggestions are also given for testing this novel strategy on real proteomic samples.