Deep Protein Methylation Profiling by Combined Chemical and Immunoaffinity Approaches Reveals Novel PRMT1 Targets(PPT)

摘要

Protein methylation has been implicated in many important biological contexts including signaling,metabolism,and transcriptional control.Despite the importance of this post-translational modification,the global analysis of protein methylation by mass spectrometry-based proteomics has not been extensively studied because of the lack of robust,well-characterized techniques for methyl peptide enrichment.Here,to better investigate protein methylation,we compared two methods for methyl peptide enrichment: immunoaffinity purification (IAP) and high pH strong cation exchange (SCX).Using both methods,we identified 1720 methylation sites on 778 proteins.Comparison of these methods revealed that they are largely orthogonal,suggesting that the usage of both techniques is required to provide a global view of protein methylation.Using both IAP and SCX,we then investigated changes in protein methylation downstream of protein arginine methyltransferase 1 (PRMT1).PRMT1 knockdown resulted in significant changes to 127 arginine methylation sites on 78 proteins.In contrast,only a single lysine methylation site was significantly changed upon PRMT1 knockdown.In PRMT1 knockdown cells,we found 114 MMA sites that were either significantly downregulated or upregulated on proteins enriched for mRNA metabolic processes.PRMT1 knockdown also induced significant changes in both asymmetric dimethyl arginine (ADMA) and symmetric dimethyl arginine (SDMA).Using characteristic neutral loss fragmentation ions,we annotated dimethylarginines as either ADMA or SDMA.Through integrative analysis of methyl forms,we identified 18 high confidence PRMT1 substrates and 12 methylation sites that are scavenged by other non-PRMT1 arginine methyltransferases in the absence of PRMT1 activity.We also identified one methylation site,HNRNPA1 R206,which switched from ADMA to SDMA upon PRMT1 knockdown.Taken together,our results suggest that deep protein methylation profiling by mass spectrometry requires orthogonal enrichment techniques to identify novel PRMT1 methylation targets and highlight the dynamic interplay between methyltransferases in mammalian cells.