Hydrophobic Derivatization Strategy Facilitates Comprehensive Profiling of Protein Methylation

Protein methylation is receiving more and more attention due to its essential role in diverse biological processes. Large-scale analysis of protein methylation requires the efficient identification of methylated peptides at the proteome level; unfortunately, a significant number of methylated peptides are highly hydrophilic and hardly retained during reversed-phase chromatography, making it difficult to be identified by conventional approaches. Herein, we report the development of a novel strategy by combining hydrophobic derivatization and high pH strong cation exchange enrichment, which significantly expands the identification coverage of the methylproteome. Noteworthily, the total number of identified methylated short peptides was improved by more than 2-fold. By this strategy, we identified 492 methylation sites from NCI-H460 cells compared to only 356 sites identified in native forms. The identification of methylation sites before and after derivatization was highly complementary. Approximately 2-fold the methylation sites were obtained by combining the results identified in both approaches (native and derivatized) as compared with the only analysis in native forms. Therefore, this novel chemical derivatization strategy is a promising approach for the comprehensive identification of protein methylation by improving the identification of methylated short peptides.