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

Abstract

Fig. S1. Gradient improvement for high pH SCX enrichment.A) Workflow of high pH SCX enrichment (1). LN229 cells were lysed in 8M urea and digested with trypsin. From 1 mg of protein, five fractions are produced by the SCX procedure and were subsequently run on a Q Exactive Plus MS. The data was searched on Proteome Discoverer 2.2 and methyl peptides were subject to a strict 1% methyl FDR. The number of PSMs for two gradients tested is shown in the table in E). B) Density plots of PSM retention times for methyl and nonmethyl PSMs run on the "Long" SCX gradient used from Wang et al.. PSMs from all 5 fractions were combined and their retention times plotted. The average density of nonmethyl PSMs and methyl PSMs was 275 PSM / min and 15 PSMs / min, respectively. C) Original "Long" gradient and a new proposed "Short" gradient for SCX. Wang et al. did not collect spectra for the first 20 min during sample loading, but because we found that methyl PSMs were eluting during this sample loading phase, we did collect spectra during this phase of the LC gradient. D) Density plot of methyl PSM retention time for methyl PSMs captured by the "Long" and "Short" SCX gradients. PSMs from all 5 fractions were combined and their retention times plotted for each gradient. The average density of "Long" and "Short" gradient methyl PSMs was 15 PSM / min and 19 PSMs / min, respectively. E) Summary of spectra identified by each gradient. Each of the five SCX fractions are shown individually. The numbers of methyl and nonmethyl PSMs were used to calculate the percent enrichment for each fraction. The number of MMA, Kme1, DMA, Kme2, PSMs, and mixed methyl PSMs are shown. Mixed PSMs contained a mixture of methyl marks on the same peptide (e.g.,MMA and DMA). The percolator q-value cutoff used to estimate the methyl FDR is also shown for each technique.

Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

“…The global identification of the substrates of PRMT1, PRMT4, and PRMT5 has been reported in several studies, thus greatly increasing our understanding of arginine methylation and its cellular functions 6 – 8 . Surprisingly, the number of putative PRMT1, PRMT4, and PRMT5 substrates was very similar 6 – 8 , 29 . Recently, Hanghandish et al have performed quantitative mass spectrometry experiments to identify PRMT7-interacting proteins, which led to the discovery of the PRMT7 direct interaction with eIF2α and its methylation 30 .…”

Improved Discrimination of Asymmetric and Symmetric Arginine Dimethylation by Optimization of the Normalized Collision Energy in Liquid Chromatography–Mass Spectrometry Proteomics

“…High pH Strong Cation Exchange (SCX) -As described previously 22,23 , 1 mg of digested protein was resuspended in loading buffer (60% acetonitrile, 40% BRUB (5 mM phosphoric acid, 5 mM boric acid, 5 mM acetic acid, pH 2.5) and incubated with high pH SCX beads (Sepax) for 30 minutes, washed with washing buffer (80% acetonitrile, 20% BRUB, pH 9), and eluted into four fractions using elution buffer 1 (60% acetonitrile, 40% BRUB, pH 9), elution buffer 2 (60% acetonitrile, 40% BRUB, pH 10), elution buffer 3 (60% acetonitrile, 40% BRUB, pH 11), and elution buffer 4 (30% acetonitrile, 70% BRUB, pH 12. Eluates were dried, resuspended in 1% trifluoroacetic acid and desalted on STAGE tips 24 with 2 mg of HLB material (Waters) loaded onto 300 uL tip with a C8 plug (Empore, Sigma).…”

A Comprehensive Analysis of the Role of hnRNP A1 Function and Dysfunction in the Pathogenesis of Neurodegenerative Disease

“…Alternatively, asymmetrical methylation of hnRNP A1 by PRTM1 at Arg206, 218, 225, and 232 has been shown to reduce IRES-mediated translation (Table 1; Wall and Lewis, 2017). When Hartel et al screened the human "methylome" by a combination of biochemical methods with and without PRTM1 knockdown, they identified a switch in methylation of hnRNP A1 Arg206, from asymmetrical to symmetrical methylation, and suggest that this may be evidence of mutually antagonistic activities of PRTM1 and PRTM5 in regulating hnRNP A1 ITAF activity (Table 1; Hartel et al, 2019).…”