A covalent conjugation enrichment strategy for analysis of dimethyllysine sites in the proteome

Lysine methylation is a common protein post-translational modification, and it has three forms as monomethyllysine (Kme1), dimethyllysine (Kme2) and trimethyllysine (Kme3). All of them have exhibited significant roles in many biological processes. Thus, development of methods for analysis of methyllysine sites is essential to further explore their biological functions. In a previous study, we found that covalent conjugation enrichment strategy has exhibited much more significant advantages in the analysis of cellular Kme1 sites than affinity-based or strong cation-exchange method. It encouraged us to develop a covalent conjugation enrichment strategy for deep exploration of Kme2 sites in the proteome. Here, we reported a Kme2 enrichment strategy including transforming Kme2 peptides into Kme1 peptides and using an aryl diazonium resin to enrich those newly produced Kme1 peptides. First, Kme2 peptides could be efficiently converted into Kme1 peptides through two steps including oxidization and elimination, and the only significant and complete side reaction on 20 natural amino acids is the oxidation on methionine. Meanwhile, propionylation could be utilized to block the original Kme1 residues for distinction with new produced ones. By this strategy, we could enrich Kme2 peptides in a complex mixture of peptides and finally identify 32 common Kme2 sites in HeLa cells by three replicates. 24 of them were unreported according to PhosphoSitePlus and 8 of them were known, including K561me2 of heat shock 70 kDa protein (HSP70), K55me2 and K165me2 of elongation factor 1-alpha 1 (EF1A1). Kme2 is an intermediate state of lysine methylation and plays significant roles in various biological processes especially on epigenetic regulation. Identification on Kme2 sites is basic for further understanding of their biological significance for both histone and nonhistone proteins. Our covalent conjugation enrichment strategy realized broad analysis of Kme2 sites in the proteome and provided great potential for exploring significant biological functions of Kme2 sites.