Global Methylation Profiling by Selective Release of Methylated Sites from Immobilized Tryptic Peptides

Methylation of lysine and arginine (K and R) has emerged as a prevalent post-translational modification with critical roles in numerous biological processes. The current identification approaches suffer from suboptimal enrichment efficiency, particularly for lysine methylation, hindering comprehensive KR methylome profiling. Herein, we presented an antibody-free strategy termed Selective Release of Methylated Sites from Immobilized Tryptic Peptides (SRMs-ITP), which achieves high enrichment efficiency while enabling the simultaneous analysis of all five methylation states of KR. This strategy exploits the unique ability of LysargiNase to cleave methylated KR residues, which are absent in trypsin-based digestion. Totally, our approach identified 5516 methylation sites across 2866 proteins from HeLa cell lysate, including 2405 arginine methylation sites and 3111 lysine methylation sites. SRMs-ITP achieved an enrichment efficiency exceeding 48.2%, significantly outperforming current antibody-based and antibody-free strategies. Notably, 56.4% of the detected methylation sites were on lysine residues, surpassing the existing antibody-free approaches. These findings establish SRMs-ITP as a robust, unbiased, and highly efficient methodology for KR methylome analysis. The approach offers a powerful tool for deciphering the intricate regulatory mechanisms of protein methylation and its cross-talk with other post-translational modifications under various physiological and pathological conditions.