Chemical derivatization of citrullinated peptides using methylglyoxal and sodium 3-mercaptopropanesulfonate (MG/MPS)

Abstract

Protein citrullination, a crucial post-translational modification, is challenging to detect due to its small mass shift and low abundance. Existing methods, including antibody-based assays and conventional chemical derivatization, often suffer from cross-reactivity, poor specificity, or multiple side reactions. To address these limitations, we developed a novel chemical derivatization strategy based on the specific interaction between sulfhydryl compounds and the ureido group of citrulline. We systematically evaluated various dicarbonyl and sulfhydryl compounds, identifying methylglyoxal (MG) and sodium 3-mercaptopropanesulfonate (MPS) as the optimal reagents for a two-step derivatization process. This method sequentially introduced +54 Da and +192 Da mass tags, enabling highly specific and sensitive detection of citrullinated peptides. Reaction conditions, including molar ratios, acid catalysts, concentration, time, and temperature, were rigorously optimized. The strategy was successfully validated using standard citrullinated peptides and a PAD4-catalyzed BSA model system, which supports its reliability for citrullinated peptide analysis and improves detection sensitivity in LC-MS/MS analysis. This MG/MPS derivatization approach provides a promising and robust technical solution for identifying protein citrullination in well-defined model systems, with the potential for further extension to complex biological environments in future studies.