(736e) Discovery of Novel Protein Methylation Targets through Unbiased, Global Methyl-Proteomics | AIChE

(736e) Discovery of Novel Protein Methylation Targets through Unbiased, Global Methyl-Proteomics


Hartel, N. - Presenter, University of Southern California
Graham, N., University of Southern California
Chew, B., USC
Xu, J., USC
Qin, J., USC
Protein arginine methylation is important for biological processes including signaling, metabolism, and transcriptional control. Despite the importance of this post-translational modification, protein methylation is relatively understudied because of its small size and neutral nature. In addition, quantitative analysis is complicated because arginine methylation occurs in three forms (monomethyl, asymmetric dimethyl, and symmetric dimethyl). Here, to improve methyl-proteomic analysis by mass spectrometry, we developed a novel method combining immunoaffinity purification (i.e., antibody-based) and chemical enrichment (i.e., strong cation exchange) of methylated peptides. Comparison revealed that these methods were largely orthogonal, suggesting that both techniques are required to provide a global view of protein methylation. Additionally, we found that label-free quantitative analysis was highly reproducible with 67% of peptides with CV below 50%. Finally, we developed a method to discriminate between the isobaric modifications asymmetric and symmetric dimethyl-arginine using characteristic neutral losses that occur during peptide sequencing. To demonstrate the power of our method, we investigated changes in protein methylation upon knockdown of the protein arginine methyltransferase 1 (PRMT1). We quantified ~1,000 methylation sites on 407 proteins and found that PRMT1 knockdown resulted in significant changes to ~90 arginine methylation sites on 59 proteins. Through integrative analysis of different methyl proteoforms, we identified 12 high confidence and 43 putative PRMT1 substrates. In addition, we identified 17 methylation sites subject to PRMT scavenging, whereby methylation sites become available to non-PRMT1 enzymes in the absence of PRMT1 activity. Our novel proteomic method demonstrates the regulatory complexity of protein arginine methylation and will enable future unbiased, quantitative studies that will highlight the importance of this understudied PTM in biological processes.


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