A chemo‐selective enrichment strategy to achieve in‐depth coverage of methyllysine proteome

Proteomics is a powerful method to comprehensively understand cellular posttranslational modifications (PTMs). Due to low abundance, tryptic peptides with PTMs are usually enriched for enhanced coverage by LC‐MS/MS. Affinity chromatography for phosphoproteomes by metal‐oxide and pan‐specific antibodies for lysine acetylome allow identification of tens of thousands of modification sites. Lysine methylation is a significant PTM, however, only hundreds of methylation sites were identified from available approaches. Here we report an aryl diazonium‐based chemoselective strategy that enables enrichment of monomethyllysine (Kme1) peptides via covalent bond with extraordinary sensitivity. We identified more than ten thousand Kme1 peptides from diverse cell lines and mouse tissues, that implied wide lysine methylation impact on cellular processes. In addition, we found a significant amount of methyl marks that were not S‐adenosyl methionine (SAM)‐dependent by isotope labeling experiments. And therefore, this method paves a way to broad application in lysine methylation research and new biology discovery.