Deep Protein Methylation Profiling by Combined Chemical and Immunoaffinity Approaches Reveals Novel PRMT1 Targets
2019; Elsevier BV; Volume: 18; Issue: 11 Linguagem: Inglês
10.1074/mcp.ra119.001625
ISSN1535-9484
AutoresNicolas Hartel, Brandon T.L. Chew, Jian Qin, Jian Xu, Nicholas A. Graham,
Tópico(s)Epigenetics and DNA Methylation
ResumoProtein 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. 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. Protein post-translational modifications (PTMs) 1The abbreviations used are:PTMpost-translational modificationADMAasymmetric dimethyl arginineDMAdimethyl arginineHILIChydrophilic interaction chromatographyIAPimmunoaffinity purificationKme1monomethyl lysineKme2dimethyl lysineKme3trimethyl lysineKMTlysine methyltransferaseLCliquid chromatographyLFQlabel-free quantitationMSmass spectrometryMMAmonomethyl argininePRMTprotein arginine methyltransferaseSCXstrong cation exchangeSDMAsymmetric dimethyl arginine. 1The abbreviations used are:PTMpost-translational modificationADMAasymmetric dimethyl arginineDMAdimethyl arginineHILIChydrophilic interaction chromatographyIAPimmunoaffinity purificationKme1monomethyl lysineKme2dimethyl lysineKme3trimethyl lysineKMTlysine methyltransferaseLCliquid chromatographyLFQlabel-free quantitationMSmass spectrometryMMAmonomethyl argininePRMTprotein arginine methyltransferaseSCXstrong cation exchangeSDMAsymmetric dimethyl arginine. regulate diverse biological processes and provide additional complexity to proteins beyond their initial primary sequence (1Beltrao P. 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App. 2000; 748: 157-166Crossref PubMed Scopus (120) Google Scholar). Through integrative analysis of MMA and DMA, we identified a list of 18 PRMT1 substrates and 12 substrates scavenged by other PRMTs in the absence of PRMT1 activity. Taken together, our results describe a general method for deep profiling of protein methylation and identify novel potential MMA and ADMA methylation targets of PRMT1. LN229 cells and HEK 293T cells expressing short hairpin RNA (shRNA) against PRMT1 or control were grown in DMEM media (Corning, Corning, NY) supplemented with 10% FBS (Omega Scientific, Tarzana, CA) and 100 U/ml penicillin/streptomycin (Thermo Scientific, Waltham, MA). Cells were cultured at 37 °C in humidified 5% CO2 atmosphere. Generation of HEK 293T cells stably expressing shRNA against PRMT1 or control were previously described (5Xu J. Wang A.H. Oses-Prieto J. Makhijani K. Katsuno Y. Pei M. Yan L. Zheng Y.G. Burlingame A. Brückner K. Derynck R. Arginine methylation initiates BMP-induced Smad signaling.Mol. Cell. 2013; 51: 5-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). shPRMT1 and shControl cells were cultured with 4 μg/ml puromycin to maintain selection. Cells were washed with PBS, scraped, and lysed in 50 mm Tris pH 7.5, 8 m urea, 1 mm activated sodium vanadate, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, and 100 mm sodium phosphate. Protein concentrations were measured by bicinchoninic assay. Lysates were sonicated and cleared by high speed centrifugation and then filtered through 0.22 μm filter. Proteins were reduced, alkylated, and quenched with 5 mm dithiothreitol, 25 mm iodoacetamide, 10 mm dithiothreitol, respectively. Lysates were 4-fold diluted in 100 mm Tris pH 8.0 and digested with trypsin at a 1:100 ratio and then quenched with addition of trifluoroacetic acid to pH 2. Peptides were purified using reverse-phase Sep-Pak C18 cartridges (Waters, Milford, MA) and eluted with 30% acetonitrile, 0.1% TFA and then dried by vacuum. Dried peptides were subjected to high pH strong cation exchange or antibody immunoaffinity purification. Cells were lysed in modified RIPA buffer (50 mm Tris-HCl (pH 7.5), 150 NaCl, 50 mm β-glycerophosphate, 0.5 mm NP-40, 0.25% sodium deoxycholate, 10 mm sodium pyrophosphate, 30 mm sodium fluoride, 2 mm EDTA, 1 mm activated sodium vanadate, 20 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm DTT, and 1 mm phenylmethylsulfonyl fluoride). Whole-cell lysates were resolved by SDS-PAGE on 4–15% gradient gels and blotted onto nitrocellulose membranes (BioRad, Hercules, CA). Membranes were blocked for 1 h in nonfat milk, and then incubated with primary and secondary antibodies overnight and for 2 h, respectively. Blots were imaged using the Odyssey Infrared Imaging System (LiCor, Lincoln, NE). Primary antibodies used for Western blot analysis were: mono-methyl arginine (8015, Cell Signaling Technology, Danvers, MA), asymmetric di-methyl arginine motif (13522, Cell Signaling), symmetric di-methyl arginine motif (13222, Cell Signaling), PRMT1 (2449, Cell Signaling), and anti-β-actin (10081–976, Proteintech, Rosemont, IL). As described previously (28Wang K. Dong M. Mao J. Wang Y. Jin Y. Ye M. Zou H. Antibody-free approach for the global analysis of protein methylation.Anal. Chem. 2016; 88: 11319-11327Crossref PubMed Scopus (31) Google Scholar), in brief, 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, Newark, DE) for 30 min, washed with washing buffer (80% acetonitrile, 20% BRUB, pH 9), and eluted into five 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), elution buffer 4 (30% acetonitrile, 70% BRUB, pH 12), and elution buffer 5 (100% BRUB, 1 m NaCl, pH 12). Eluates were dried, resuspended in 1% trifluoroacetic acid and desalted on STAGE tips (35Rappsilber J. Ishihama Y. Mann M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.Anal. Chem. 2003; 75: 663-670Crossref PubMed Scopus (1796) Google Scholar) with 2 mg of HLB material (Waters) loaded onto 300 μl tip with a C8 plug (Empore, Sigma, St. Louis, MO). Ten milligrams of digested proteins were dissolved in 1× immunoprecipitation buffer (50 mm MOPS, 10 mm Na2HPO4, 50 mm NaCl, pH 7.2, Cell Signaling). Modified symmetric dimethyl arginine peptides, asymmetric dimethyl arginine peptides, and monomethyl arginine peptides were immunoprecipitated by addition of 40 μl of PTMScan Symmetric Di-Methyl Arginine Motif Kit (13563, Cell Signaling), PTMScan Asymmetric Di-Methyl Arginine Motif Kit (13474, Cell Signaling), and PTMScan Mono-Methyl Arginine Motif Kit (12235, Cell Signaling), respectively. Modified methyl lysine peptides were enriched with PTMScan Pan-Methyl Lysine Kit (14809). Lysates were incubated with PTMScan motif kits for 2 h at 4 °C on a rotator. Beads were centrifuged and washed two times in 1X immunoprecipitation buffer followed by three washes in water, and modified peptides were eluted with 2 × 50 μl of 0.15% TFA and desalted on STAGE tips with C18 cores (Empore, Sigma). Enriched peptides were resuspended in 50 mm ammonium bicarbonate (Sigma) and subjected to a second digestion with trypsin for 2 h per the manufacturer's recommendation, acidified with trifluoroacetic acid to pH 2 and desalted on STAGE tips. All LC-MS experiments were performed on a nanoscale UHPLC system (EASY-nLC1200, Thermo Scientific) connected to an Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer equipped with a nanoelectrospray source (Thermo Scientific). Peptides were separated by a reversed-phase analytical column (PepMap RSLC C18, 2 μm, 100 Å, 75 μm × 25 cm) (Thermo Scientific). For high pH SCX fractions a "Short" gradient was used where flow rate was set to 300 nl/min at a gradient starting with 0% buffer B (0.1% FA, 80% acetonitrile) to 29% B in 142 min, then washed by 90% B in 10 min, and held at 90% B for 3. The maximum pressure was set to 1,180 bar and column temperature was constant at 50 °C. For IAP samples a "Slow" gradient was used where flow rate was set to 300 nl/min at a gradient starting with 0% buffer B to 25% B in 132 min, then washed by 90% B in 10 min. Dried SCX fractions were resuspended in buffer A and injected as follows, E1: 1.5 μl/60 μl, E2–5: 5 μl/6 μl. IAP samples were resuspended in 7 μl and 6.5 μl was injected. The effluent from the HPLC was directly electrosprayed into the mass spectrometer. Peptides separated by the column were ionized at 2.0 kV in the positive ion mode. MS1 survey scans for DDA were acquired at resolution of 70k from 350 to 1,800 m/z, with maximum injection time of 100 ms and AGC target of 1e6. MS/MS fragmentation of the 10 most abundant ions were analyzed at a resolution of 17.5k, AGC target 5e4, maximum injection time 120 ms for IAP samples, 240 ms for SCX samples, and normalized collision energy 26. Dynamic exclusion was set to 30 s and ions with charge 1 and >6 were excluded. MS/MS fragmentation spectra were searched with Proteome Discoverer SEQUEST (version 2.2, Thermo Scientific) against the in-silico tryptic digested Uniprot H. sapiens database with all reviewed with isoforms (release Jun 2017, 42,140 entries). The maximum missed cleavage rate was set to 5 (28Wang K. Dong M. Mao J. Wang Y. Jin Y. Ye M. Zou H. Antibody-free approach for the global analysis of protein methylation.Anal. Chem. 2016; 88: 11319-11327Crossref PubMed Scopus (31) Google Scholar). Trypsin was set to cleave at R and K. Dynamic modifications were set to include mono-methylation of arginine or lysine (R/K, +14.01565), di-methylation of arginine or lysine (R/K, +28.0313), tri-methylation of lysine (K, +42.04695), oxidation on methionine (M, +15.995 Da, and acetylation on protein N terminus (+42.011 Da). Fixed modification was set to carbamidomethylation on cysteine residues (C, +57.021 Da). The maximum parental mass error was set to 10 ppm and the MS/MS mass tolerance was set to 0.02 Da. Peptides with sequence of six to fifty amino acids were considered. Methylation site localization was determined by ptm-RS node in Proteome Discoverer, and only sites with localization probability greater or equal to 75% were considered. The False Discovery Rate threshold was set strictly to 0.01 using Percolator node validated by q-value. Relative abundances of parental peptides were calculated by integration of area-under-the-curve of the MS1 peaks using Minora LFQ node in Proteome Discoverer 2.2. The Proteome Discoverer export peptide groups abundance values were log2 transformed, normalized to the corresponding samples median values, and significance was determined using a permutation-based FDR approach in the Perseus environment (37Tyanova S. Temu T. Sinitcyn P. Carlson A. Hein M.Y. Geiger T. Mann M. Cox J. The Perseus computational platform for comprehensive analysis of (prote)omics data.Nat. Methods. 2016; 13: 731-740Crossref PubMed Scopus (3530) Google Scholar) (release 1.6.2.3) with a q-value FDR of 0.05 and S0 value of 0.5. The "Decoy PSMs" export from Proteome Discoverer 2.2 was filtered for decoy methyl PSMs and the decoy q-values from the Percolator node were extracted and compared with the target methyl PSM q-values. Target methyl PSMs were removed until a 1% FDR was achieved as described (38Elias J.E. Gygi S.P. Target-decoy search strategy for mass spectrometry-based proteomics.Methods Mol. Biol. Clifton NJ. 2010; 604: 55-71Crossref PubMed Scopus (404) Google Scholar). The modifications SDMA and ADMA were added to MaxQuant's library with the added mass of dimethyl on arginine and the corresponding neutral loss masses of 31.042 for SDMA and 45.058 for ADMA assigned in the "Neutral Loss" Table in Configuration (26Musiani D. Bok J. Massignani E. Wu L. Tabaglio T. Ippolito M.R. Cuomo A. Ozbek U. Zorgati H. Ghoshdastider U. Robinson R.C. Guccione E. Bonaldi T. Proteomics profiling of arginine methylation defines PRMT5 substrate specificity.Sci Signal. 2019; 12: eaat8388Crossref PubMed Scopus (74) Google Scholar). The missed cleavage rate was set to 5 and all other settings were kept unchanged. All RAW files were searched with monomethyl(K/R), ADMA, SDMA, and oxidation of methionine as variable modifications. Carbamidomethylation was kept as a fixed modification. Neutral losses and their masses were extracted from the msms.txt file. Only target methyl peptides that passed the 1% Methyl FDR filter were considered for analysis. An Andromeda cutoff score of 56 was also used to filter spectra to reduce the number of incorrect assignments. A custom R script was used to remove neutral losses that did not have the corresponding b/y ion present (e.g. if y6* but not y6 was present, the neutral loss was removed). A few spectra were confirmed by manual inspection to ensure the accuracy of the Andromeda search. For identified ADMA/SDMA neutral losses, the Andromeda output was matched to Proteome Discoverer data by MS2 scan number. Motifs were analyzed by MotifX (39Schwartz D. Chou M.F. Church G.M. Predicting protein post-translational modifications using meta-analysis of proteome scale data sets.Mol. Cell. Proteomics MCP. 2009; 8: 365-379Abstract Full Text Full Text PDF PubMed Scopus (89) Goo
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