Strategy Based on Deglycosylation, Multiprotease, and Hydrophilic Interaction Chromatography for Large-Scale Profiling of Protein Methylation

Reversible methylation of proteins regulates the majority of cellular processes, including signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation. A fundamental understanding of these biological processes at the molecular level requires comprehensive characterization of the methylated proteins. Methylation is often substoichiometric, and only a very limited number of methylated proteins and sites have been confidently identified to date. Although the intrinsically basic/hydrophilic methylated peptides can be enriched by the hydrophilic interaction liquid chromatography (HILIC), other hydrophilic peptides can coelute during the enrichment process and suppress the detection of methylated peptides. In addition, the modified Arg and Lys residues cannot be efficiently cleaved by trypsin, the most commonly used enzyme in shotgun proteomics. To overcome these caveats, we develop a novel de-glyco-assisted methylation site identification (DOMAIN) strategy which enables straightforward, fast, and reproducible analysis of protein methylation in a proteome-wide manner. Combining multidimensional fractionation and multiprotease digestion, our method enabled the identification of 573 methylated forms in 270 proteins, including 311 new methylation forms, in A549 cells. Combining this technique with stable isotope labeling quantitative proteomics and RNA interference, we determined the differential regulation of several putative methylated sites that are related to the protein arginine <i>N</i>-methyltransferase 3 (PRMT3). Collectively, our integrated proteomics workflow for comprehensive mapping of methylation sites enables a better understanding of protein methylation, while providing a rapid and effective approach for global protein methylation analysis in biomedical research