Enrichment of Methylated Peptides Using an Antibody-free Approach for Global Methylproteomics Analysis

Protein methylation is receiving increasing attention for its important role in regulating diverse biological processes, including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Proteome level analysis of protein methylation requires the enrichment of various forms of methylated peptides. Unfortunately, immunoaffinity purification can only enrich a subset of them due to the lack of pan-specific antibodies. Chromatography-based methods, however, can enrich methylated peptides in a global manner. Here we present a chromatography-based approach for highly efficient enrichment of methylated peptides. Protocols for the of high pH SCXtip preparation and methyl-peptide purification are described in detail. Key points such as cell culture in hM-SILAC medium and protein digestion by multiple endopeptidases are also presented. This technique allows the simultaneous analysis of both lysine and arginine methylation and improved performance for methyl-arginine identification. © 2018 by John Wiley & Sons, Inc.