Global Proteomics Analysis of Protein Lysine Methylation

Xing‐Jun Cao1, Benjamin A. Garcia1

1 Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.8
DOI:  10.1002/cpps.16
Online Posting Date:  November, 2016
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Abstract

Lysine methylation is a common protein post‐translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non‐histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non‐histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites. However, recently, there has been a series of publications to perform large‐scale analysis of protein lysine methylation. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. © 2016 by John Wiley & Sons, Inc.

Keywords: immunoaffinity enrichment; methylation; mass spectrometry; non‐histone; post‐translational modification; proteomics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Large‐Scale Analysis of Protein Lysine Methylation in Cells
  • Support Protocol 1: Antisera Generation and Affinity Purification of Antibody
  • Support Protocol 2: Preparation of Capillary Columns with Integrated Emitter Tips
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Large‐Scale Analysis of Protein Lysine Methylation in Cells

  Materials
  • 0.25% trypsin‐EDTA (Gibco)
  • 1 M Tris·HCl, pH 8.3 (see recipe)
  • Urea lysis buffer (see recipe)
  • Bradford protein assay solution (see unit 3.4; Olson and Markwell, )
  • 1 M DTT stock solution (see recipe)
  • 1 M iodoacetamide (IAA) solution (see recipe)
  • Trypsin
  • Trifluoroacetic acid (TFA)
  • Methanol, HPLC grade
  • Acetic acid
  • Formic acid (FA)
  • Acetonitrile
  • SCX Buffer A (see recipe)
  • SCX Buffer B (see recipe)
  • Protein A Mag sepharose (GE Healthcare)
  • Pan anti‐monomethyl lysine antibody (see protocol 2)
  • Saturated Na 2HPO 4 in PBS (see recipe)
  • 0.8 M NaCl in PBS
  • Reverse‐phase HPLC Buffer A (see recipe)
  • Reverse‐phase HPLC Buffer B (see recipe)
  • pH indicator strip (pH range 1 to 14)
  • Centrifuge
  • Sep‐Pak C18 cartridges (Waters)
  • PolySULFOETHYL A column (9.4 mm I.D. × 250 mm, PolyLC)
  • Empore C18 disk (3M)
  • Reverse‐phase HPLC column (see protocol 3)
  • Probe sonicator
  • QIAvac vacuum manifold
  • HPLC with UV‐detector
  • SpeedVac concentrator
  • Magnetic rack
  • Tube rotator

Support Protocol 1: Antisera Generation and Affinity Purification of Antibody

  Additional Materials (also see protocol 1Basic Protocol)
  • Tris(2‐carboxyethyl) phosphine (TCEP)
  • 1 M NaCl
  • 50 mM L‐cysteine (see recipe)
  • SulfoLink coupling resin
  • Resin coupling buffer (see recipe)
  • Sodium azide
  • Centrifuge
  • Econo‐Pac chromatography columns
  • Tube rotator

Support Protocol 2: Preparation of Capillary Columns with Integrated Emitter Tips

  Materials
  • Methanol
  • Acetonitrile
  • HF acid
  • 0.2 M ammonium formate
  • Reprosil‐Pur C18‐AQ resin (3 µm; Dr. Maisch)
  • BSA digests
  • Reverse‐phase HPLC Buffer A (see recipe)
  • 360 μm O.D. × 75 μm I.D. fused silica tubing
  • Lighter
  • Lamp
  • Laser puller P‐2000
  • Helium tank and its accessories
  • Pressure bomb (Nanobaume, cat. no. SP‐400)
  • 2‐ml HPLC glass vial
  • Stirrer
  • Microscope
  • HPLC instrument (e.g., Easy nLC 1000 HPLC)
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Figures

Videos

Literature Cited

Literature Cited
  Autiero, M., Luttun, A., Tjwa, M., and Carmeliet, P. 2003. Placental growth factor and its receptor, vascular endothelial growth factor receptor‐1: Novel targets for stimulation of ischemic tissue revascularization and inhibition of angiogenic and inflammatory disorders. J. Thromb. Haemost. 1:1356‐1370. doi: 10.1046/j.1538‐7836.2003.00263.x.
  Beausoleil, S.A., Villen, J., Gerber, S.A., Rush, J., and Gygi, S.P. 2006. A probability‐based approach for high‐throughput protein phosphorylation analysis and site localization. Nat. Biotechnol. 24:1285‐1292. doi: 10.1038/nbt1240.
  Bremang, M., Cuomo, A., Agresta, A.M., Stugiewicz, M., Spadotto, V., and Bonaldi, T. 2013. Mass spectrometry‐based identification and characterisation of lysine and arginine methylation in the human proteome. Mol. Biosyst. 9:2231‐2247. doi: 10.1039/c3mb00009e.
  Cao, X.J., Arnaudo, A.M., and Garcia, B.A. 2013. Large‐scale global identification of protein lysine methylation in vivo. Epigenetics 8:477‐485. doi: 10.4161/epi.24547.
  Chi, H., He, K., Yang, B., Chen, Z., Sun, R.X., Fan, S.B., Zhang, K., Liu, C., Yuan, Z.F., Wang, Q.H., Liu, S.Q., Dong, M.Q., and He, S.M. 2015. pFind‐Alioth: A novel unrestricted database search algorithm to improve the interpretation of high‐resolution MS/MS data. J. Proteomics 125:89‐97. doi: 10.1016/j.jprot.2015.05.009.
  Choudhary, C., Kumar, C., Gnad, F., Nielsen, M.L., Rehman, M., Walther, T.C., Olsen, J.V., and Mann, M. 2009. Lysine acetylation targets protein complexes and co‐regulates major cellular functions. Science 325:834‐840. doi: 10.1126/science.1175371.
  Chuikov, S., Kurash, J.K., Wilson, J.R., Xiao, B., Justin, N., Ivanov, G.S., McKinney, K., Tempst, P., Prives, C., Gamblin, S.J., Barlev, N.A., and Reinberg, D. 2004. Regulation of p53 activity through lysine methylation. Nature 432:353‐360. doi: 10.1038/nature03117.
  Copeland, R.A., Solomon, M.E., and Richon, V.M. 2009. Protein methyltransferases as a target class for drug discovery. Nat. Rev. Drug Discov. 8:724‐732. doi: 10.1038/nrd2974.
  Cox, J. and Mann, M. 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.‐range mass accuracies and proteome‐wide protein quantification. Nat. Biotechnol. 26:1367‐1372. doi: 10.1038/nbt.1511.
  Guo, A., Gu, H., Zhou, J., Mulhern, D., Wang, Y., Lee, K.A., Yang, V., Aguiar, M., Kornhauser, J., Jia, X., Ren, J., Beausoleil, S.A., Silva, J.C., Vemulapalli, V., Bedford, M.T., and Comb, M.J. 2014. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation. Mol. Cell Proteomics 13:372‐387. doi: 10.1074/mcp.O113.027870.
  Hamamoto, R., Saloura, V., and Nakamura, Y. 2015. Critical roles of non‐histone protein lysine methylation in human tumorigenesis. Nat. Rev. Cancer 15:110‐124. doi: 10.1038/nrc3884.
  Hojfeldt, J.W., Agger, K., and Helin, K. 2013. Histone lysine demethylases as targets for anticancer therapy. Nat. Rev. Drug Discov. 12:917‐930. doi: 10.1038/nrd4154.
  Huang, J., Perez‐Burgos, L., Placek, B.J., Sengupta, R., Richter, M., Dorsey, J.A., Kubicek, S., Opravil, S., Jenuwein, T., and Berger, S.L. 2006. Repression of p53 activity by Smyd2‐mediated methylation. Nature 444:629‐632. doi: 10.1038/nature05287.
  Kim, W., Bennett, E.J., Huttlin, E.L., Guo, A., Li, J., Possemato, A., Sowa, M.E., Rad, R., Rush, J., Comb, M.J., Harper, J.W., and Gygi, S.P. 2011. Systematic and quantitative assessment of the ubiquitin‐modified proteome. Mol. Cell 44:325‐340. doi: 10.1016/j.molcel.2011.08.025.
  Lundby, A., Lage, K., Weinert, B.T., Bekker‐Jensen, D.B., Secher, A., Skovgaard, T., Kelstrup, C.D., Dmytriyev, A., Choudhary, C., Lundby, C., and Olsen, J.V. 2012. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns. Cell Rep. 2:419‐431. doi: 10.1016/j.celrep.2012.07.006.
  Mazur, P.K., Reynoird, N., Khatri, P., Jansen, P.W., Wilkinson, A.W., Liu, S., Barbash, O., Van Aller, G.S., Huddleston, M., Dhanak, D., Tummino, P.J., Kruger, R.G., Garcia, B.A., Butte, A.J., Vermeulen, M., Sage, J., and Gozani, O. 2014. SMYD3 links lysine methylation of MAP3K2 to Ras‐driven cancer. Nature 510:283‐287. doi: 10.1038/nature13320.
  Mertins, P., Qiao, J.W., Patel, J., Udeshi, N.D., Clauser, K.R., Mani, D.R., Burgess, M.W., Gillette, M.A., Jaffe, J.D., and Carr, S.A. 2013. Integrated proteomic analysis of post‐translational modifications by serial enrichment. Nat. Methods 10:634‐637. doi: 10.1038/nmeth.2518.
  Moore, K.E., Carlson, S.M., Camp, N.D., Cheung, P., James, R.G., Chua, K.F., Wolf‐Yadlin, A., and Gozani, O. 2013. A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation. Mol. Cell 50:444‐456. doi: 10.1016/j.molcel.2013.03.005.
  Nakakido, M., Deng, Z., Suzuki, T., Dohmae, N., Nakamura, Y., and Hamamoto, R. 2015. Dysregulation of AKT pathway by SMYD2‐mediated lysine methylation on PTEN. Neoplasia 17:367‐373. doi: 10.1016/j.neo.2015.03.002.
  Ong, S.‐E. and Mann, M. 2006. Identifying and quantifying sites of protein methylation by heavy methyl SILAC. Curr. Protoc. Protein Sci. 46:14.9.1‐14.9.12. doi: 10.1002/0471140864.ps1409s46.
  Ong, S.‐E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell Proteomics 1:376‐386. doi: 10.1074/mcp.M200025‐MCP200.
  Ong, S.‐E., Mittler, G., and Mann, M. 2004. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat. Methods 1:119‐126. doi: 10.1038/nmeth715.
  Olson, B.J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4.1‐3.4.29. doi: 10.1002/0471140864.ps0304s48.
  Pang, C.N., Gasteiger, E., and Wilkins, M.R. 2010. Identification of arginine‐ and lysine‐methylation in the proteome of Saccharomyces cerevisiae and its functional implications. BMC Genomics 11:92. doi: 10.1186/1471‐2164‐11‐92.
  Piao, L., Kang, D., Suzuki, T., Masuda, A., Dohmae, N., Nakamura, Y., and Hamamoto, R. 2014. The histone methyltransferase SMYD2 methylates PARP1 and promotes poly(ADP‐ribosyl)ation activity in cancer cells. Neoplasia 16:257‐264. doi: 10.1016/j.neo.2014.03.002.
  Rappsilber, J., Ishihama, Y., and Mann, M. 2003. Stop and go extraction tips for matrix‐assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75:663‐670. doi: 10.1021/ac026117i.
  Rotili, D. and Mai, A. 2011. Targeting histone demethylases: A new avenue for the fight against cancer. Genes Cancer 2:663‐679. doi: 10.1177/1947601911417976.
  Saddic, L.A., West, L.E., Aslanian, A., Yates, J.R. III, Rubin, S.M., Gozani, O., and Sage, J. 2010. Methylation of the retinoblastoma tumor suppressor by SMYD2. J. Biol. Chem. 285:37733‐37740. doi: 10.1074/jbc.M110.137612.
  Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J.R., Cole, P.A., Casero, R.A., and Shi, Y. 2004. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119:941‐953. doi: 10.1016/j.cell.2004.12.012.
  Tan, M., Peng, C., Anderson, K.A., Chhoy, P., Xie, Z., Dai, L., Park, J., Chen, Y., Huang, H., Zhang, Y., Ro, J., Wagner, G.R., Green, M.F., Madsen, A.S., Schmiesing, J., Peterson, B.S., Xu, G., Ilkayeva, O.R., Muehlbauer, M.J., Braulke, T., Muhlhausen, C., Backos, D.S., Olsen, C.A., McGuire, P.J., Pletcher, S.D., Lombard, D.B., Hirschey, M.D., and Zhao, Y. 2014. Lysine glutarylation is a protein posttranslational modification regulated by SIRT5. Cell Metab. 19:605‐617. doi: 10.1016/j.cmet.2014.03.014.
  Wagner, T. and Jung, M. 2012. New lysine methyltransferase drug targets in cancer. Nat. Biotechnol. 30:622‐623. doi: 10.1038/nbt.2300.
  Wu, Z., Cheng, Z., Sun, M., Wan, X., Liu, P., He, T., Tan, M., and Zhao, Y. 2015. A chemical proteomics approach for global analysis of lysine monomethylome profiling. Mol. Cell Proteomics 14:329‐339. doi: 10.1074/mcp.M114.044255.
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