Enrichment and Identification of Methylation at the Proteome Level

Abstract

Methylation is a post-translational modification which occurs on lysine and arginine residues. Methylation is difficult to detect due to its low abundance and lack of charge. Our laboratory previously developed a novel enrichment approach, ProMENADe, for lysine and arginine methylation in the human embryonic kidney (HEK) 293T cell line which is coupled with mass spectrometry. Simplifying a lysate with subcellular fractionation prior to enrichment increased the identification of methylation sites by 39.5% while using multiple proteases for digestion increased identification by 27%. Combining these methods yielded a 47.2% increase. Analysis at the 1% methylation level FDR filtered for C-terminal methylation identified 169 sites and further analysis revealed 74 of these sites overlap with the PhosphoSite database. This ProMENADe enrichment strategy yielded 95 novel methylation sites to the field and can be a key tool in the field of methylation allowing for the enrichment and identification of methylated proteins.