An efficient approach based on basic strong cation exchange chromatography for enriching methylated peptides with high specificity for methylproteomics analysis

Abstract

Protein methylation as one of the most important post-translational modifications has been under the spotlight due to its essential role in many biological processes. Development of methods for large-scale analysis of protein methylation greatly accelerates the related researches. To date, antibody-based enrichment strategy is the most common approach for methylproteomics analysis. However, it is still lacking of a pan-specific antibody to enrich peptides or proteins carrying all kinds of lysine and arginine methylation forms. Herein, an online basic strong cation exchange chromatography was developed to enrich methylated peptides from protein digests prepared by two complementary methods, including direct multiple enzymes digestion and carboxylic amidation followed by multiple enzymes digestion. After enrichment, the majority of identifications were obtained from direct multiple enzymes digested sample. The enrichment specificity of methylated peptides was up to 28.5%, and 445 methylation forms corresponding to 376 methylation sites were identified on 194 proteins in one LC-MS/MS run using only 100 μg of digests. This method has great potential in studying protein methylation mediated biological processes.