Global lysine methylome profiling using systematically characterized affinity reagents.

Area of Science:

• Biochemistry
• Proteomics
• Molecular Biology

Background:

• Lysine methylation regulates protein function and is implicated in various human diseases.
• Identifying lysine methylation sites is essential for understanding the roles of lysine methyltransferases (KMTs) and lysine demethylases (KDMs).
• Current mass spectrometry (MS)-based detection methods rely on pan-methyllysine antibodies, which may have limitations.

Purpose of the Study:

• To investigate the sequence bias of commercially available pan-methyllysine antibodies.
• To evaluate the impact of antibody selectivity on MS-based detection of methylated peptides.
• To improve the comprehensive identification of lysine methylation sites in the human proteome.

Main Methods:

• Peptide microarrays were used to assess the in vitro sequence bias of pan-methyllysine antibodies.
• Multiple enrichment strategies were employed in MS-based workflows.
• Global lysine methylation proteomics was performed on two human cell lines.

Main Results:

• Most commercially available pan-methyllysine antibodies exhibit significant in vitro sequence bias.
• Utilizing multiple enrichment approaches enhances the coverage of the lysine methylome.
• The study identified 5089 lysine methylation sites on 2751 proteins, nearly doubling the previously reported sites.

Conclusions:

• Pan-methyllysine antibody selectivity can bias the detection of lysine methylation.
• Employing diverse enrichment methods is critical for comprehensive lysine methylome analysis.
• This work significantly expands the catalog of identified lysine methylation sites, aiding future research into their functional roles and disease relevance.