Utilizing a Negative Enrichment Strategy to Profile Protein Methylation, Leveraging the Orthogonality of LysargiNase and Trypsin.

Protein methylation, a prevalent post-translational modification, plays crucial roles in chromatin remodeling and gene transcription. A deeper understanding of protein methylation in these biological processes requires comprehensive characterization of the methylation sites. However, methylation induces minimal changes in the size and electrostatic status of lysine/arginine residues, especially in the case of mono-methylation and dimethylation. This significantly increases the difficulty in distinguishing methylation sites from non-methylation sites. In this study, we developed a strategy to enrich protein methylation, termed the Negative Enrichment Strategy for Profiling Protein Methylation, to comprehensively analyze lysine/arginine methylation. Initially, proteins were digested using LysargNase to generate peptides containing methylated or non-methylated lysine/arginine at the N-terminus. Subsequently, the N-terminal free α-amines of the LysargiNase-generated peptides were selectively blocked using formaldehyde in an acidic solution. Since trypsin cleaves after non-methylated lysine/arginine residues, only non-methylated peptides were digested by trypsin, exposing neo-N-terminal free amines. Finally, the non-methylated peptides with neo-N-terminal free amines were selectively removed by reacting with hyperbranched polyglycerol-aldehyde polymers, resulting in the negative enrichment of methylated peptides. Through our approach, we identified 2419 methylation forms in 2384 sites from 1440 proteins. This method provided a powerful approach for the comprehensive profiling of protein lysine and arginine methylations simultaneously, enabling a deeper understanding of protein methylation in diverse cellular conditions and human diseases.