Deep protein methylation profiling by combined chemical and immunoaffinity approaches reveals novel PRMT1 targets

Abstract

Running title: Deep methylation profiling reveals novel PRMT1 targetsDeep methylation profiling reveals novel PRMT1 targets 2 ABBREVIATIONS ADMA, asymmetric dimethyl arginine; DMA, dimethyl arginine; HILIC, hydrophilic interaction chromatography; IAP, immunoaffinity purification; Kme1, monomethyl lysine; Kme2, dimethyl lysine; Kme3, trimethyl lysine; KMT, lysine methyltransferase; LC, liquid chromatography; LFQ, label-free quantitation; MS, mass spectrometry; MMA, monomethyl arginine; PTM, posttranslational modification; PRMT, protein arginine methyltransferase; SCX, strong cation exchange; SDMA, symmetric dimethyl arginine;Deep methylation profiling reveals novel PRMT1 targets 3 ABSTRACT 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 due to 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 1,720 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. Deep methylation profiling reveals novel PRMT1 targets

Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function

“…To filter out target peptide spectrum matches over the decoy-peptide spectrum matches, a fixed FDR of 1% was set at the peptide level. Mass recalibration and label free quantification (LFQ) of area under the curve MS1 peaks were carried out using Proteome Discoverer nodes, Spectrum Files RC, Minora Feature Detector and Feature Mapper (31)(32)(33)(34)(35)(36)(37).…”

Proteome-wide identification of arginine methylation in colorectal cancer tissues from patients

“…Using immuneenrichment of arginine-methylated peptides combined with MS-based proteomics technology, thousands of arginine-methylated sites have been identified from various biological sources [8][9][10][11]. Furthermore, this technology can be available for site-specific quantitative characterization of methylarginines between control and experimental groups [12,13]. As new enrichment strategy for arginine/lysine-methylated peptides has been developed [14], it is expected that high resolution MS technique combined with efficient enrichement strategies of arginine-methylated pepties become more valuable for both basic science and biomedical research.…”

Extracellular 5′-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins

“…Effluent from the HPLC was then directly electrosprayed into the mass spectrometer. The parameters for the MS/MS analysis was essentially identical to that described ( 34 ). MS/MS fragmentation spectra were searched with MaxQuant software ( 35 ) (version 1.6.0.1) using Andromeda 1.5.6.0 against the Swiss-Prot human protein database (downloaded on April 24, 2019; 20,402 entries).…”