Detecting Methylation in MCF7 Cells with a Combination of SCX- and No-Enrichment Methods

Abstract Methylation is an important post-translational modification that impacts cellular processes. Yet, protein methylation remains analytically challenging to study by proteomics due to variable methylation stoichiometry and the lack of broadly applicable enrichment strategies, resulting in an underrepresentation of methylated peptides in LC-MS/MS analyses. Although methylation does not alter the physicochemical properties of amino acids, there is a reduced probability of trypsin cleavage after methylated lysine or arginine, leading to increased positive charges on methylated peptides. Strong cation exchange (SCX) can be used to enrich methylated peptides. However, the number of methylation sites identified with SCX varies across previous studies, likely because of the use of different stationary phases and supports, elution strategies, and combinations of multiple enrichment techniques. Therefore, we hypothesize that commercially available SCX tips with an MS-friendly ammonium acetate elution can be used to enrich methylated peptides. Our results show that SCX effectively fractionates peptides by charge, which is associated with the number of missed cleavages, and that methylated peptides elute in fractions with high concentrations of ammonium acetate. When comparing SCX and nonenrichment methods, complementary methylation sites are identified, differing by sequence motifs, cellular component ontologies, and biological process ontologies. Ultimately, these results suggest that both SCX and nonenrichment methods enable the detection of distinct methylation sites, and a combination of approaches may be required to effectively profile the methylome of complex biological samples. Data have been deposited in MassIVE (MSV000098142).