Reversible methylation of proteins regulates the majority of cellular processes, 3 including signal transduction, mRNA splicing, transcriptional control, DNA repair, and 4 protein translocation. A fundamental understanding of these biological processes at the 5 molecular level requires comprehensive characterization of the methylated proteins. 6 Methylation is often substoichiometric, and only a very limited number of methylated 7 proteins and sites have been confidently identified to date. Although the intrinsically 8 basic/hydrophilic methylated peptides can be enriched by the hydrophilic interaction 9 liquid chromatography (HILIC), other hydrophilic peptides can co-elute during the 10 enrichment process and suppress the detection of methylated peptides. In addition, the 11 modified Arg and Lys residues cannot be efficiently cleaved by trypsin, the most 12 commonly used enzyme in shotgun proteomics. To overcome these caveats, we develop a 13 novel De-glycO-assisted MethylAtion site IdeNtification (DOMAIN) strategy which 14 enables straightforward, fast, and reproducible analysis of protein methylation in a 15 proteome-wide manner. Combining multi-dimensional fractionation and multi-protease 16 digestion, our method enabled the identification of 573 methylated forms in 270 proteins, 17 including 311 new methylation forms, in A549 cells. Combining this technique with 18 stable isotope labeling quantitative proteomics and RNA interference, we determined the 19 differential regulation of several putative methylated sites that are related to protein 20 arginine N-methyltransferase 3 (PRMT3). Collectively, our integrated proteomics 21 workflow for comprehensive mapping of methylation sites enables a better understanding Page 2 of 28 Analytical Chemistry of protein methylation, while providing a rapid and effective approach for global protein 2 methylation analysis in biomedical research