Middle-down proteomics reveals RNA binding proteins harbor dense sites of methylation and phosphorylation

Arginine (Arg)-rich RNA-binding proteins play an integral role in RNA metabolism. Post-translational modifications (PTMs) within Arg-rich domains, such as phosphorylation and methylation, regulate multiple steps in RNA metabolism. However, the identification of PTMs within Arg-rich domains with complete trypsin digestion is extremely challenging due to the high density of Arg residues within these proteins. Here, we leveraged a middle-down proteomic approach coupled with electron transfer dissociation (ETD) mass spectrometry to map previously unknown sites of phosphorylation and methylation within the Arg-rich domains of U1-70K and structurally similar RNA binding proteins. From nuclear extracts of HEK293 cells, we achieved coverage of 29,114 residues and identified 681 PTMs currently unannotated in database repositories. Remarkably, the Arg-rich domains in RNA binding proteins are densely modified by methylation and phosphorylation compared with the remainder of the proteome, with methylation and phosphorylation often jointly occurring in Arg-rich peptides within RSRS motifs. Although they share a common motif, phosphorylation and methylation may oppose one another. Collectively, these findings suggest that the level of PTMs within Arg-rich domains may be among the highest in the proteome, and a possible unexplored regulator of RNA metabolism. These data also serve as a resource to facilitate future mechanistic studies of these PTMs in RNA binding protein structure and function.