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A Method for Large-scale Identification of Protein Arginine Methylation

2012; Elsevier BV; Volume: 11; Issue: 11 Linguagem: Inglês

10.1074/mcp.m112.020743

1535-9484

Thomas Uhlmann, Vincent Geoghegan, Benjamin Thomas, Gabriela Ridlova, David C. Trudgian, Oreste Acuto,

Peptidase Inhibition and Analysis

The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel proline-rich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking. The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel proline-rich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking. Knowledge of the type, extent and dynamics of post-translational modifications (PTMs) 1The abbreviations used are:PTMspost-translational modificationsMeth-Rmethylated argininesADMAasymmetric dimethylarginineGARglycine-arginine-richSDMAsymmetric dimethylarginineMMAmonomethylargininePRMTsprotein arginine methyltransferasesHILIChydrophilic interaction liquid chromatographySCXstrong cation exchangeSILACstable isotope labeling by amino acids in cell culturePRAMproline-rich arginine methylationFDRfalse discovery rate.1The abbreviations used are:PTMspost-translational modificationsMeth-Rmethylated argininesADMAasymmetric dimethylarginineGARglycine-arginine-richSDMAsymmetric dimethylarginineMMAmonomethylargininePRMTsprotein arginine methyltransferasesHILIChydrophilic interaction liquid chromatographySCXstrong cation exchangeSILACstable isotope labeling by amino acids in cell culturePRAMproline-rich arginine methylationFDRfalse discovery rate. reveals the changing network of protein interactions and the regulation of cellular functions. Methylated arginines (Meth-R) are relatively frequent in cellular proteins (e.g. 0.7–1% of total arginines (1Bulau P. Zakrzewicz D. Kitowska K. Wardega B. Kreuder J. Eickelberg O. Quantitative assessment of arginine methylation in free versus protein-incorporated amino acids in vitro and in vivo using protein hydrolysis and high-performance liquid chromatography.BioTechniques. 2006; 40: 305-310Crossref PubMed Scopus (32) Google Scholar) and this work) and have a very slow turnover (2Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar), likely to confer lasting functional properties to proteins. Meth-Rs are often found at glycine-arginine-rich (GAR) sequences and modulate protein-protein and protein-nucleic acid interactions by reducing hydrogen-bonding and local hydrophilicity (2Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar). Methylation at arginine can weaken interactions but also enhance the binding of adaptors called Tudor domains to Meth-R-containing sequences (3Sprangers R. Groves M.R. Sinning I. Sattler M. High-resolution X-ray and NMR structures of the SMN Tudor domain: conformational variation in the binding site for symmetrically dimethylated arginine residues.J. Mol. Biol. 2003; 327: 507-520Crossref PubMed Scopus (142) Google Scholar, 4Yang Y. Lu Y. Espejo A. Wu J. Xu W. Liang S. Bedford M.T. TDRD3 is an effector molecule for arginine-methylated histone marks.Mol. Cell. 2010; 40: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). post-translational modifications methylated arginines asymmetric dimethylarginine glycine-arginine-rich symmetric dimethylarginine monomethylarginine protein arginine methyltransferases hydrophilic interaction liquid chromatography strong cation exchange stable isotope labeling by amino acids in cell culture proline-rich arginine methylation false discovery rate. post-translational modifications methylated arginines asymmetric dimethylarginine glycine-arginine-rich symmetric dimethylarginine monomethylarginine protein arginine methyltransferases hydrophilic interaction liquid chromatography strong cation exchange stable isotope labeling by amino acids in cell culture proline-rich arginine methylation false discovery rate. Meth-R occurs in three forms: asymmetric dimethylarginine (ADMA), bearing two methyl groups on one nitrogen of the guanidino group; symmetric dimethylarginine (SDMA), where both nitrogens are singly methylated and monomethylarginine (MMA), a reaction intermediate. In humans, these reactions are catalyzed by a family of protein arginine methyltransferases (PRMTs), with PRMT1, 3, 4, 6, and 8 forming ADMA and PRMT5 and 7 producing SDMA. In mice, PRMT1 and PRMT4 deficiency causes embryonic (5Yu Z. Chen T. Hébert J. Li E. Richard S. A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation.Mol. Cell. Biol. 2009; 29: 2982-2996Crossref PubMed Scopus (141) Google Scholar) or Perinatal (6Kim J. Lee J. Yadav N. Wu Q. Carter C. Richard S. Richie E. Bedford M.T. Loss of CARM1 results in hypomethylation of thymocyte cyclic AMP-regulated phosphoprotein and deregulated early T cell development.J. Biol. 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The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation.PLoS Biol. 2006; 4: e355Crossref PubMed Scopus (163) Google Scholar, 12Zhao Q. Rank G. Tan Y.T. Li H. Moritz R.L. Simpson R.J. Cerruti L. Curtis D.J. Patel D.J. Allis C.D. Cunningham J.M. Jane S.M. PRMT5-mediated methylation of histone H4R3 recruits DNMT3A, coupling histone and DNA methylation in gene silencing.Nat. Struct. Mol. Biol. 2009; 16: 304-311Crossref PubMed Scopus (394) Google Scholar) and tune the activity of transcription factors (2Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar, 13Zhao X. Jankovic V. Gural A. Huang G. Pardanani A. Menendez S. Zhang J. Dunne R. Xiao A. Erdjument-Bromage H. Allis C.D. Tempst P. Nimer S.D. Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity.Genes Dev. 2008; 22: 640-653Crossref PubMed Scopus (137) Google Scholar, 14Jansson M. Durant S.T. Cho E.C. Sheahan S. Edelmann M. Kessler B. La Thangue N.B. Arginine methylation regulates the p53 response.Nat. Cell Biol. 2008; 10: 1431-1439Crossref PubMed Scopus (350) Google Scholar, 15Covic M. Hassa P.O. Saccani S. Buerki C. Meier N.I. Lombardi C. Imhof R. Bedford M.T. Natoli G. Hottiger M.O. Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression.EMBO J. 2005; 24: 85-96Crossref PubMed Scopus (181) Google Scholar), coactivators (16Chen D. Ma H. Hong H. Koh S.S. Huang S.M. Schurter B.T. Aswad D.W. Stallcup M.R. Regulation of transcription by a protein methyltransferase.Science. 1999; 284: 2174-2177Crossref PubMed Scopus (1000) Google Scholar, 17Fathman J.W. Gurish M.F. Hemmers S. Bonham K. Friend D.S. Grusby M.J. Glimcher L.H. Mowen K.A. NIP45 controls the magnitude of the type 2 T helper cell response.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 3663-3668Crossref PubMed Scopus (21) Google Scholar) and corepressors (8Ancelin K. Lange U.C. Hajkova P. Schneider R. Bannister A.J. Kouzarides T. Surani M.A. Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells.Nat. Cell Biol. 2006; 8: 623-630Crossref PubMed Scopus (376) Google Scholar, 11Jelinic P. Stehle J.C. Shaw P. The testis-specific factor CTCFL cooperates with the protein methyltransferase PRMT7 in H19 imprinting control region methylation.PLoS Biol. 2006; 4: e355Crossref PubMed Scopus (163) Google Scholar). Factors involved in mRNA splicing (18Liu Q. Dreyfuss G. In vivo and in vitro arginine methylation of RNA-binding proteins.Mol. Cell. Biol. 1995; 15: 2800-2808Crossref PubMed Scopus (268) Google Scholar, 19Ohkura N. Takahashi M. Yaguchi H. Nagamura Y. Tsukada T. 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Cell. 2007; 25: 71-83Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) often contain Meth-R, suspected to be involved in protein-RNA interaction (2Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar). Moreover, T cell CD28 co-stimulation (22Blanchet F. Cardona A. Letimier F.A. Hershfield M.S. Acuto O. CD28 costimulatory signal induces protein arginine methylation in T cells.J. Exp. Med. 2005; 202: 371-377Crossref PubMed Scopus (80) Google Scholar), TNFα (15Covic M. Hassa P.O. Saccani S. Buerki C. Meier N.I. Lombardi C. Imhof R. Bedford M.T. Natoli G. Hottiger M.O. Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression.EMBO J. 2005; 24: 85-96Crossref PubMed Scopus (181) Google Scholar), TLR4 (15Covic M. Hassa P.O. Saccani S. Buerki C. Meier N.I. Lombardi C. Imhof R. Bedford M.T. Natoli G. Hottiger M.O. Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression.EMBO J. 2005; 24: 85-96Crossref PubMed Scopus (181) Google Scholar), NGF (23Cimato T.R. Tang J. Xu Y. Guarnaccia C. Herschman H.R. Pongor S. Aletta J.M. Nerve growth factor-mediated increases in protein methylation occur predominantly at type I arginine methylation sites and involve protein arginine methyltransferase 1.J. Neurosci. Res. 2002; 67: 435-442Crossref PubMed Scopus (62) Google Scholar) and NFkB (15Covic M. Hassa P.O. Saccani S. Buerki C. Meier N.I. Lombardi C. Imhof R. Bedford M.T. Natoli G. Hottiger M.O. Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression.EMBO J. 2005; 24: 85-96Crossref PubMed Scopus (181) Google Scholar) signaling pathways may be regulated by PRMTs. Protein arginine methylation is implicated in pathogenic processes, including oncogenesis (24Cheung N. Chan L.C. Thompson A. Cleary M.L. So C.W. Protein arginine-methyltransferase-dependent oncogenesis.Nat. Cell. Biol. 2007; 9: 1208-1215Crossref PubMed Scopus (249) Google Scholar), cardiovascular disease (25Wang Z. Tang W.H. Cho L. Brennan D.M. Hazen S.L. Targeted metabolomic evaluation of arginine methylation and cardiovascular risks: potential mechanisms beyond nitric oxide synthase inhibition.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1383-1391Crossref PubMed Scopus (111) Google Scholar), autoimmunity and viral infections (26Bedford M.T. Richard S. Arginine methylation an emerging regulator of protein function.Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar), raising the possibility that abnormally methylated proteins can be disease markers and PRMTs potential therapeutic targets (27Copeland R.A. Solomon M.E. Richon V.M. Protein methyltransferases as a target class for drug discovery.Nat. Rev. Drug discovery. 2009; 8: 724-732Crossref PubMed Scopus (376) Google Scholar). Only a very limited number of arginine methylated proteins and sites have been identified with certainty to date (see UniProtKB). In vitro elegant screenings were developed to search for PRMT substrates (21Cheng D. Côté J. Shaaban S. Bedford M.T. The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing.Mol. Cell. 2007; 25: 71-83Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) but identifying the Meth-R concerned is usually cumbersome. The existence of Meth-R sites is in many cases only suspected by protein similarity such as the presence of GAR motifs, which are substrates for some but not all PRMTs (2Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1250) Google Scholar). These vexing limitations hamper progress of a very promising field in biology. Previous work developed a powerful approach using heavy methyl-SILAC coupled to MS to identify with high certainty peptides containing Meth-R (28Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (365) Google Scholar). An anti-ADMA antibody was used to enrich for Meth-R-containing proteins but only a limited number of Meth-R were identified (28Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (365) Google Scholar). The relative low abundance of Meth-R-containing proteins and peptides and lack of adequate methods for their enrichment remains therefore a major hurdle for both large- and small-scale discovery of Meth-R sites, leaving the heavy methyl-SILAC method essentially unexploited. Here, we have uncovered distinct physico-chemical properties of Meth-R-containing tryptic peptides and showed that hydrophilic interaction liquid chromatography (HILIC) is a simple method providing excellent enrichment of Meth-R-containing peptides. When coupled to heavy methyl-stable isotope labeling by amino acids in cell culture (SILAC) based MS, HILIC allowed us to identify hundreds of Meth-R sites, providing for the first time a means for "methylome"-scale investigation that vastly outperforms the use of anti-ADMA antibody. Custom-made RPMI 1640 medium lacking l-Methionine, l-Arginine, and l-Lysine (Invitrogen, Carlsbad, CA) was supplemented with 10% dialyzed fetal calf serum (Invitrogen), l-Arginine and l-Lysine and either a) l-Methionine (CK Gas Products Ltd.), or b) l-Methionine-methyl-13C-methyl-D3 (Sigma, ISOTECTM) at a final concentration of 0.1 mm l-Methionine, 0.29 mm l-Arginine and 0.219 mm l-Lysine and filter sterilized (0.22 μm pore size, Millipore, Billerica, MA). Jurkat cells were grown at 37 °C in a humidified 5% CO2-containing atmosphere for 5–7 cell doublings in labeling media. Human primary CD4+ lymphocytes were isolated from whole blood of healthy donors by negative selection using a Dynal isolation kit (Invitrogen) according to the manufacturer's protocol. 1 × 106 to 2 × 106 CD4+ lymphocytes in 10 ml of light or heavy medium were then stimulated by plate bound anti-CD3 at 10 μg/ml and soluble CD28.2 (BioLegend, San Diego, CA) at 1 μg/ml. IL-2 (AbDSerotec) was added to a concentration of 50 ng/ml. Cells were grown at 37 °C in a humidified 5% CO2-containing atmosphere for 7–9 days. Cells were harvested and lysed for either a) 10 min in ice-cold lysis buffer (20 mm Tris pH 7.5, 150 mm NaCl, 0.1 mm EDTA, 1% Nonidet P-40, 0.5% Deoxicholate, protease inhibitor mixture (Roche), or b) 20 min in 8 m Urea in 25 mm Tris pH 8. Lysates were cleared by centrifugation at 14,000 × g for 20 min and mixed in a 1:1 ratio of protein content (measured by Bradford method and absorbance at 280 nm). Lysates were used for a) immunoprecipitation, or b) strong Cation exchange (SCX) chromatography, HILIC and OFFGEL isoelectric focusing. Immunoprecipitation was performed on the mixed cell lysates overnight at 4 °C on Sepharose protein G beads (GE Healthcare) pre-incubated with an anti-DMA mAb 7E6 (Abcam, Cambridge, UK). Beads were washed four times with fresh ice-cold lysis buffer and eluted with 100 mm triethylamine pH 11.5 for 10 min at RT. The eluates were neutralized with 1/20 volume of 1 m phosphate buffer pH 6.8, boiled in reducing SDS NuPAGE sample buffer (Invitrogen), alkylated with 55 mm iodoacteamide (Sigma) and separated on 4–12% gradient Bis-Tris NuPAGE gels (Invitrogen). The gel was washed with distilled water and lightly stained with Colloidal Blue (Invitrogen). The gel lane was divided into 15 slices and subject to GeLCMS/MS. Cleared Urea lysate was acetone precipitated and resuspended in 1.6 m Urea in 25 mm Tris pH8. Overnight digestion at 37 °C was carried out with 12.5 ng/μl trypsin (Proteomics grade, Sigma) or chymotrypsin (Sequencing grade, Promega, Madison, WI). For SCX, 4–6 mg peptides were acidified with 0.1% trifluoroacetic acid (Sigma) and cleared by centrifuging at 20,000 × g for 10 min. Peptides were applied to a 1 ml SCX column (Resource S, GE Healthcare) using Buffer A (50 mm KH2PO4 20% acetonitrile, pH2.7). Bound peptides were eluted with increasing Buffer B (50 mm KH2PO4, 20% acetonitrile, 1 m KCl, pH2.7). In a first region of separation the gradient was stepped up to 7% Buffer B to elute 1+ and most of the 2+ charged peptides. A shallow gradient to 35% Buffer B separated the remaining 2+ from 3+ and higher charged peptides. In the last stage the Buffer B was changed to pH7 and a steep gradient to 100% Buffer B ensured complete elution of bound peptides. Following C18 desalting (Empore Octadecyl C18, Sigma), the fractions were analyzed by LC-MS/MS. For HILIC 1 mg of peptides were acidified with 0.1% trifluoroacetic acid (TFA) and cleared by centrifuging at 20,000g for 10 min. Peptides were loaded onto a reverse phase C2/C18 column (GE Healthcare) with Buffer A (0.1% formic acid, pH2.7). Elution of peptides was carried out with a steep gradient to 100% Buffer B (0.1% formic acid, 95% acetonitrile, pH2.7). Desalted peptides were then dried under vacuum, re-suspended in Buffer B and applied to a HILIC column (Merck). Bound peptides were eluted with a shallow gradient of Buffer A up to 80%. For IEF on OFFGEL, 1 mg of peptides were loaded onto an IPG strip (13 cm pH7–11, GE Healthcare) with IPG buffer (pH7–11, GE Healthcare) according to manufacturer's instructions. Peptides were focused in solution with an Agilent 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA) for 20 kVh up to a maximum voltage of 8000 V. Focused peptides were removed and desalted with C18 (Empore Octadecyl C18, Sigma). Peptides were also extracted by immersing the paper wicks from the cathode end in 0.1% TFA for 1–2 h, followed by brief sonication. Peptides from the respective fractions were collected and adjusted to 0.1% TFA, 5% acetonitrile and applied to a homemade C18 stage tip for desalting. Bound peptides were washed with 0.1% TFA, 2% acetonitrile and eluted in 0.1% TFA, 60% acetonitrile. Eluted peptides were dried and sent to the PNAC facility in Cambridge (http://www.bioc.cam.ac.uk/pnac/aaa.hml) for amino acid analysis on a Biochrome 30 Analyzer. Samples were analyzed on an Ultimate 3000 nano HPLC (Dionex, Camberley, UK) system run in direct injection mode coupled to either a LTQ XL Orbitrap mass spectrometer, or a Q Exactive mass spectrometer (Thermo Electron, Hemel Hempstead, UK). Samples were resolved on a 15 cm by 75 μm inner diameter picotip analytical column (New Objective, Woburn, MA), which was packed in-house with Reprosil-Pur C18-AQ phase, 3 μm bead (Dr. Maisch, Germany). A 120 min gradient was used to separate the peptides and each sample was typically injected three times and data merged in order to increase sample coverage. The LTQ XL Orbitrap mass spectrometer was operated in a "Top 5" data dependent acquisition mode and the Q Exactive mass spectrometer was operated in a "Top 10" data dependent acquisition mode. Precursor scans were performed at a resolving power of 60,000, from which either five precursor ions (LTQ XL Orbitrap) or 10 precursor ions (Q Exactive) were selected and fragmented. Charge state +1 ions were rejected. MS/MS peak lists were converted to mzXML format using ReAdW version 4.4.1 (LTQ XL Orbitrap data) or to mgf format using ProteoWizard msconvert release 3.0.3535 (Q Exactive data). Both tools were used with default parameters except that zlib compression of spectral data was enabled. Data was uploaded to the central proteomics facilities pipeline (CPFP at: https://cpfp-master.molbiol.ox.ac.uk/cpfp_demo/auth/login) (29Trudgian D.C. Thomas B. McGowan S.J. Kessler B.M. Salek M. Acuto O. CPFP: a central proteomics facilities pipeline.Bioinformatics. 2010; 26: 1131-1132Crossref PubMed Scopus (76) Google Scholar). Files were searched using Mascot version 2.3.01 (Matrix science), X!TANDEM version 2008.12.01.1 (30Craig R. Beavis R.C. TANDEM: matching proteins with tandem mass spectra.Bioinformatics. 2004; 20: 1466-1467Crossref PubMed Scopus (1987) Google Scholar) and OMSSA version 2.1.8 (31Geer L.Y. Markey S.P. Kowalak J.A. Wagner L. Xu M. Maynard D.M. Yang X. Shi W. Bryant S.H. Open mass spectrometry search algorithm.J. Proteome Res. 2004; 3: 958-964Crossref PubMed Scopus (1164) Google Scholar) against a concatenated target and reversed decoy version of the IPI human sequence database (EBI), versions 3.78 and 3.79 containing 86,702 and 86,635 target protein sequences respectively. Enzyme was set to trypsin (or chymotrypsin for some experiments) allowing for up to 3 missed cleavages. Carbamidomethyl cysteine was set as a fixed modification. For analysis of arginine methylation oxidized methionine, heavy oxidized methionine, monomethylarginine, heavy monomethylarginine, dimethylarginine, and heavy dimethylarginine were set as variable modifications. For analysis of lysine methylation, oxidized methionine, heavy oxidized methionine, monomethyllysine, heavy monomethyllysine, dimethyllysine, heavy dimethyllysine, trimethyllysine, and heavy trimethyllysine were set as variable modifications. Mass tolerances for MS and MS/MS peak identifications were 10 ppm and 0.5 Da respectively (LTQ XL Orbitrap) or 20 ppm and 0.1 Da respectively (Q Exactive). Mass spectrometry data are available at http://www.peptideatlas.org/PASS/PASS00081. Assignment of a methylation site required identification by searches in the Central proteomics facility pipeline (CPFP) at 1% FDR and the presence of a confirming peptide in the precursor spectrum of equal intensity separated by a mass difference introduced by the light/heavy methyl group(s). The actual false positive rate of identification using the heavy methyl SILAC criteria (1.2%, supplemental Fig. S5) was calculated on a representative dataset by analyzing all false positive hits for arginine methylation for the presence of confirming heavy methyl SILAC pairs. This number was then doubled and divided by the total number of hits for arginine methylation. The requirement for the presence of a methylSILAC pair to corroborate each methylated peptide increases confidence and enables even low scoring peptides to be considered (28Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (365) Google Scholar). Modification localization scoring was performed using the ModLS algorithm (unpublished 2David C. Trudgian, Rachelle Singleton, Matthew E. Cockman, Peter J. Ratcliffe, and Benedikt M. Kessler, ModLS: Post-translational modification localization scoring with automatic specificity expansion 2012 (submitted).) which extends the PTMScore method to incorporate automatic specificity expansion. For each variable modification chosen for the database search all amino acid specificities defined in the Unimod database (www.unimod.org) are considered during localization. This allows the correct localization of e.g. dimethylation to lysine, even if only arginine methylation was chosen for the search. Ambiguous modification localizations are annotated as such in supplemental Table S5. Within CPFP, inference of protein identifications from peptide sequences was performed using ProteinProphet (32Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3621) Google Scholar). Default parameters were used for protein grouping and the assignment of isoform identifications. Precursor m/z, charge, InterProphet probability for all methylated peptides found in this work are listed in supplemental Table S4. Biosynthetic labeling with methionine (M0) and [13CD3]-methionine (M4) (heavy methyl-SILAC) (Fig. 1A) enables detection of protein methylation sites by LC-MS/MS with high confidence (28Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (365) Google Scholar). In this study, we labeled Jurkat T cells by heavy methyl-SILAC and extended this approach to primary cells by showing efficient SILAC labeling of antigen receptor stimulated T-cells (supplemental Figs. S1, S2). In a previous study, enrichment of Meth-R-containing proteins from Hela cells with the monoclonal antibody (mAb) 7E6 to MMA and ADMA led to the identification of 59 Meth-R sites (28Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat. Methods. 2004; 1: 119-126Crossref PubMed Scopus (365) Google Scholar). By a similar approach (see Methods), we identified 41 Meth-R sites in Jurkat T cells, only 6 of which were novel (e.g. not annotated in the UniProtKB database) (supplemental Table S1). However, cell proteomes should comprise higher numbers of Meth-R-containing proteins, since 0.7–1.0% of arginine in proteins extracted from cell lines or tissues is methylated (1Bulau P. Zakrzewicz D. Kitowska K. Wardega B. Kreuder J. Eickelberg O. Quantitative assessment of arginine methylation in free versus protein-incorporated amino acids in vitro and in vivo using protein hydrolysis and high-performance liquid chromatography.BioTechniques. 2006; 40: 305-310Crossref PubMed Scopus (32) Google Scholar) (and our unpublished data). Moreover, similar to previous investigations (28Ong S.E. Mittler G. Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.Nat.