Phosphoproteomics

Jun Zhong1, Henrik Molina1, Akhilesh Pandey1

1 Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.4
DOI:  10.1002/0471140864.ps2404s50
Online Posting Date:  November, 2007
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Abstract

Protein phosphorylation is one of the most important mechanisms of regulating protein function in cells, and it plays an important role in controlling diverse biological processes, including cellular proliferation, migration, and metabolism. The term “phosphoproteome” refers to the complement of proteins that undergoes phosphorylation, the extent of their phosphorylation status at the level of individual residues, as well as the dynamics of the phosphorylation events in response to various stimuli. This unit provides methods for enrichment of phosphorylated proteins and peptides using anti‐phosphotyrosine antibodies or titanium dioxide, respectively. Support protocols are provided for two detergent‐free cell lysis methods, fractionation of proteins prior to enrichment, and use of stable isotope labeling by amino acids in cell culture (SILAC) method for studying dynamics of phosphorylation events. Curr. Protoc. Protein Sci. 50:24.4.1‐24.4.21. © 2007 by John Wiley & Sons, Inc.

Keywords: signal transduction; kinase; substrate; mass spectrometry; quantitative proteomics; bioinformatics

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Enrichment of Tyrosine‐Phosphorylated Proteins Using Anti‐Phosphotyrosine Antibodies
  • Basic Protocol 2: Enrichment of Phosphopeptides Using Titanium Dioxide
  • Support Protocol 1: Cell Lysis Using Detergent‐Free Sonication
  • Support Protocol 2: Cell Lysis Using Nitrogen Decompression (Nitrogen Cavitation)
  • Support Protocol 3: Fractionation of Proteins Using RP‐HPLC
  • Support Protocol 4: Differential Labeling of Cells Using SILAC for Relative Quantitation of the Phosphoproteome
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enrichment of Tyrosine‐Phosphorylated Proteins Using Anti‐Phosphotyrosine Antibodies

  Materials
  • Cells of interest, in culture (e.g., see appendix 3C)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Cell lysis buffer (see recipe), ice cold
  • Protein A–agarose beads (Sigma or Pierce)
  • Anti‐phosphotyrosine mouse monoclonal antibody: clone 4G10, agarose‐conjugated (Upstate Biotechnology) and clone PY20, agarose‐conjugated (BIOMOL International)
  • 100 mM phenyl phosphate (Sigma‐Aldrich) in PBS (see appendix 2E): prepare fresh just before use
  • 500‐ml centrifuge bottles
  • 15‐ml conical tubes
  • Probe‐tip sonicator (e.g., Sonifier, Branson)
  • 50‐ml tubes
  • Test tube rotator
  • Dialysis tubing (molecular weight cut off: 3,500 Da)
  • Vacuum centrifuge: e.g., Vacufuge (Eppendorf) or SpeedVac (Thermo Scientific)
  • Additional reagents and equipment for performing SDS‐PAGE (unit 10.1), Coomassie blue staining (unit 10.5), and in‐gel protein digestion (unit 11.3)

Basic Protocol 2: Enrichment of Phosphopeptides Using Titanium Dioxide

  Materials
  • 20‐ to 30‐µm titanium dioxide particles (Glygen)
  • TiO 2 suspension solution (see recipe)
  • Dried desalted (see unit 24.3, protocol 8) peptides from a proteolytic digest (in‐gel digestion, see unit 11.3; in‐solution digestion, see unit 11.1)
  • Peptide loading solution (see recipe)
  • 3% (w/v) ammonium hydroxide
  • Empore C8 membrane disk (3M Bioanalytical Technologies)
  • 10‐µl GeLoader tips (Eppendorf)
  • Vacuum centrifuge: e.g., Vacufuge (Eppendorf) or SpeedVac (Thermo Scientific)

Support Protocol 1: Cell Lysis Using Detergent‐Free Sonication

  Materials
  • Cells of interest, in culture (e.g., see appendix 3C)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Cell suspension buffer (see recipe)
  • 70% (v/v) ethanol
  • 15‐ml centrifuge tube
  • Probe‐tip sonicator (e.g., Sonifier, Branson)
  • 50‐ml centrifuge tube
  • Additional reagents and equipment for determining cell density ( appendix 3C)

Support Protocol 2: Cell Lysis Using Nitrogen Decompression (Nitrogen Cavitation)

  Materials
  • 70% (v/v) ethanol
  • Cells of interest, in culture (e.g., see appendix 3C)
  • Cell suspension buffer (see recipe)
  • Cell disruption bomb (Parr Instrument)
  • Nitrogen filling connection (Parr Instrument)
  • High‐pressure nitrogen gas tank (>1500 psi; e.g., Airgas)
  • 50‐ml centrifuge tube
  • Additional reagents and equipment for determining protein concentration (unit 3.4)

Support Protocol 3: Fractionation of Proteins Using RP‐HPLC

  Materials
  • Urea
  • 500 µg proteins from detergent‐free cell lysate ( protocol 3 or protocol 42)
  • 100 mM ammonium bicarbonate
  • 1 M dithiothreitol (freshly prepared)
  • 600 mM iodoacetamide (freshly prepared)
  • Phosphate‐buffered saline (PBS; see appendix 2E)
  • Protein standards (e.g., 20 µg bovine serum albumin undergoing the same processing as the samples)
  • Trifluoroacetic acid (TFA)
  • HPLC‐grade acetonitrile
  • Trypsin, proteomic grade
  • 20 mM ammonium bicarbonate
  • 50‐ml test tubes
  • mRP‐C18 (4.6 mm × 50 mm) high‐recovery protein column (Agilent Technologies)
  • 96‐well plate (1 ml per well; Agilent Technologies)
  • Vacuum centrifuge: e.g., Vacufuge (Eppendorf) or SpeedVac (Thermo Scientific)
  • Additional reagents and equipment for measuring protein concentration (unit 3.4) and performing HPLC (unit 8.7)

Support Protocol 4: Differential Labeling of Cells Using SILAC for Relative Quantitation of the Phosphoproteome

  Materials
  • Stable isotope–labeled L‐lysine and L‐arginine (13C 6‐L‐lysine and 13C 6‐L‐arginine for two‐state SILAC experiment; Cambridge Isotope Laboratories or Sigma‐Aldrich)
  • Customized SILAC labeling medium: cell culture medium deficient in L‐lysine and L‐arginine (Invitrogen or AthenaES)
  • Supplements required for complete medium: e.g., fetal bovine serum (Invitrogen), dialyzed (optional)
  • Cell culture medium (normal medium containing the naturally abundant isotopic forms of amino acids)
  • Cells of interest, in culture (e.g., see appendix 3C)
  • 0.2‐µm sterilization filter unit and cell culture–grade sterile filter bottle
  • 50‐ml centrifuge tube
  • Additional reagents and equipment for measuring total protein concentration (unit 3.4)
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Figures

Videos

Literature Cited

   Amanchy, R., Kalume, D.E., Iwahori, A., Zhong, J., and Pandey, A. 2005. Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC). J. Proteome Res. 4:1661‐1671.
   Amoresano, A., Marino, G., Cirulli, C., and Quemeneur, E. 2004. Mapping phosphorylation sites: A new strategy based on the use of isotopically labelled DTT and mass spectrometry. Eur. J. Mass Spectrom. 10:401‐412.
   Ballif, B.A., Villen, J., Beausoleil, S.A., Schwartz, D., and Gygi, S.P. 2004. Phosphoproteomic analysis of the developing mouse brain. Mol. Cell. Proteomics 3:1093‐1101.
   Blagoev, B., Ong, S.E., Kratchmarova, I., and Mann, M. 2004. Temporal analysis of phosphotyrosine‐dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 22:1139‐1145.
   Bodenmiller, B., Mueller, L.N., Mueller, M., Domon, B., and Aebersold, R. 2007. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat. Methods 4:231‐237.
   Cao, P. and Stults, J.T. 2000. Mapping the phosphorylation sites of proteins using on‐line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry. Rapid Commun. Mass Spectrom. 14:1600‐1606.
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   Diella, F., Cameron, S., Gemund, C., Linding, R., Via, A., Kuster, B., Sicheritz‐Ponten, T., Blom, N., and Gibson, T.J. 2004. Phospho.ELM: A database of experimentally verified phosphorylation sites in eukaryotic proteins. BMC Bio‐ informatics 5:79.
   Ficarro, S.B., McCleland, M.L., Stukenberg, P.T., Burke, D.J., Ross, M.M., Shabanowitz, J., Hunt, D.F., and White, F.M. 2002. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20:301‐305.
   Gerber, S.A., Rush, J., Stemman, O., Kirschner, M.W., and Gygi, S.P. 2003. Absolute quantitation of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. U.S.A. 100:6940‐6945.
   Goshe, M.B., Conrads, T.P., Panisko, E.A., Angell, N.H., Veenstra, T.D., and Smith, R.D. 2001. Phosphoprotein isotope‐coded affinity tag approach for isolating and quantitating phosphopeptides in proteome‐wide analyses. Anal. Chem. 73:2578‐2586.
   Gronborg, M., Kristiansen, T.Z., Stensballe, A., Andersen, J.S., Ohara, O., Mann, M., Jensen, O.N., and Pandey, A. 2002. A mass spectrometry‐based proteomic approach for identification of serine/threonine‐phosphorylated proteins by enrichment with phospho‐specific antibodies: Identification of a novel protein, Frigg, as a protein kinase A substrate. Mol. Cell. Proteomics. 1:517‐527.
   Gruhler, A., Olsen, J.V., Mohammed, S., Mortensen, P., Faergeman, N.J., Mann, M., and Jensen, O.N. 2005. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol. Cell. Proteomics 4:310‐327.
   Hornbeck, P.V., Chabra, I., Kornhauser, J.M., Skrzypek, E., and Zhang, B. 2004. PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics 4:1551‐1561.
   Kane, S., Sano, H., Liu, S.C., Asara, J.M., Lane, W.S., Garner, C.C., and Lienhard, G.E. 2002. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase‐activating protein (GAP) domain. J. Biol. Chem. 277:22115‐22118.
   Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P., and Jorgensen, T.J. 2005. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 4:873‐886.
   Molina, H., Horn, D.M., Tang, N., Mathivanan, S., and Pandey, A. 2007. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 104:2199‐2204.
   Oda, Y., Nagasu, T., and Chait, B.T. 2001. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19:379‐382.
   Olsen, J.V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., and Mann, M. 2006. Global, in vivo, and site‐specific phosphorylation dynamics in signaling networks. Cell 127:635‐648.
   Peri, S., Navarro, J.D., Amanchy, R., Kristiansen, T.Z., Jonnalagadda, C.K., Surendranath, V., Niranjan, V., Muthusamy, B., Gandhi, T.K., Gronborg, M., Ibarrola, N., Deshpande, N., Shanker, K., Shivashankar, H.N., Rashmi, B.P., Ramya, M.A., Zhao, Z., Chandrika, K.N., Padma, N., Harsha, H.C., Yatish, A.J., Kavitha, M.P., Menezes, M., Choudhury, D.R., Suresh, S., Ghosh, N., Saravana, R., Chandran, S., Krishna, S., Joy, M., Anand, S.K., Madavan, V., Joseph, A., Wong, G.W., Schiemann, W.P., Constantinescu, S.N., Huang, L., Khosravi‐Far, R., Steen, H., Tewari, M., Ghaffari, S., Blobe, G.C., Dang, C.V., Garcia, J.G., Pevsner, J., Jensen, O.N., Roepstorff, P., Deshpande, K.S., Chinnaiyan, A.M., Hamosh, A., Chakravarti, A., and Pandey, A. 2003. Development of human protein reference database as an initial platform for approaching systems biology in humans. Genome Res. 13:2363‐2371.
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Internet Resources
   http://www.silac.org
  This regularly updated Web site provides additional literature, reagents, recipes and applications for the SILAC method.
   http://www.hprd.org
  Three regularly updated Web sites provide information about phosphorylation sites. See Table for a description of their features.
   http://phospho.elm.eu.org
   http://www.phosphosite.org
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