Preparation and Analysis of Phosphorylated Proteins

Jeffrey N. Siegel1

1 Naval Medical Research Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.2
DOI:  10.1002/0471142735.im1102s03
Online Posting Date:  May, 2001
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The function of cells of the immune system is regulated in part by engagement of specific receptors on the cell surface by soluble ligands or ligands presented on the surface of other cells. One of the major mechanisms by which cell‐surface receptor engagement influences cellular function is by activating protein kinases and increasing the phosphorylation of critical cellular proteins. There are two major categories of protein kinases: serine/threonine kinases which phosphorylate both serine and threonine residues; and tyrosine kinases which phosphorylate tyrosine residues exclusively. By metabolically labeling cells with 32P, as described in this unit, the phosphorylation state of cellular proteins can be analyzed and changes occurring with receptor stimulation can be examined. Phosphoamino acid analysis is also described to determine whether serine, threonine, or tyrosine residues are phosphorylated, thereby indicating which category of protein kinase is responsible. A protocol for phosphopeptide mapping is provided to allow the investigator to produce a “fingerprint” of the peptides that are phosphorylated, providing information regarding the sites of phosphorylation.

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Table of Contents

  • Basic Protocol 1: [32P]Orthophosphate Labeling of Cells
  • Basic Protocol 2: Phosphoamino Acid Analysis
  • Basic Protocol 3: Two‐Dimensional Phosphopeptide Mapping
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: [32P]Orthophosphate Labeling of Cells

  • Cells for labeling
  • Phosphate free–RPMI 1640 (GIBCO/BRL #320‐1870AJ), without and with recipedialyzed 10% FCS (see reagents and solutions; Biofluids)
  • 400 to 800 mCi/ml [32P]orthophosphate in 0.02 N HCl (ICN Biomedicals)
  • recipePBS with phosphatase inhibitors (PBS/PI), ice cold
  • recipeLysis buffer, ice cold
  • 1× and 2× SDS/sample buffer (unit 8.4)
  • recipeGel fixation solution
  • 3% glycerol in H 2O
  • 25‐, 75‐, or 150‐cm2 tissue culture flasks (e.g., Nunc, Fisher)
  • Protective benchtop beta shield (see critical parameters and troubleshooting; Nalge, Apple Scientific #258243)
  • Plexiglass or acrylic vial beta shield (see critical parameters and troubleshooting; Nalge #66715‐0010, Apple Scientific #258244)
  • Transfer pipets ( Becton Dickinson)
  • Plexiglass or acrylic ⅜ in. thick storage box for transporting labeled cells ( Nalge #6740‐1108, Apple Scientific #253‐418)
  • 50‐ and 15‐ml screw‐cap tubes ( Sarstedt), sterile
  • Additional reagents and equipment for determining the number of cells using trypan blue exclusion ( appendix 3A), immunoprecipitation (unit 8.3), SDS‐PAGE (unit 8.4), and autoradiography ( appendix 3A)

Basic Protocol 2: Phosphoamino Acid Analysis

  • Methanol
  • Electroblotting buffer (unit 8.10)
  • Polyacrylamide gel containing labeled proteins (first protocol 1basic protocol)
  • recipeRadioactive ink
  • 5.7 N HCl
  • recipepH 1.9 and recipepH 3.5 buffers, ice cold
  • Phosphoamino acid (PAA) standards
  • recipe1% (w/v) ninhydrin spray
  • 0.45 µm‐pore‐size Immobilon‐P membrane (Millipore)
  • Whatman 3MM filter paper
  • Flatbed electrophoresis unit (Multiphor II system, Pharmacia)
  • 20 × 20–cm cellulose, glass‐backed TLC plate ( EM Science)
  • Wicks for thin‐layer electrophoresis consisting of plastic‐backed absorbent paper (“diapers”) cut to 10 × 20 cm
  • Additional reagents and equipment for immunoblotting (unit 8.10) and autoradiography ( appendix 3A)

Basic Protocol 3: Two‐Dimensional Phosphopeptide Mapping

  • 1.5% (w/v) polyvinylpyrrolidone 40 (PVP 40; Sigma) in 100 mM acetic acid
  • 95:5 (v/v) 0.05 M ammonium bicarbonate (Sigma)/acetonitrile (Baker reagent grade, VWR Scientific)
  • 2 mg/ml TPCK‐trypsin ( Worthington) in 0.05 M ammonium bicarbonate (Sigma)
  • Performic acid (Baker reagent grade, VWR Scientific), ice cold (optional)
  • recipeMarker dye
  • recipepH 8.9or recipepH 4.72 buffer
  • Chromatography buffer
  • Polyacrylamide gel containing labeled proteins (first protocol 1basic protocol)
  • 0.2‐µM‐pore‐size nitrocellulose membrane (Schleicher & Schuell)
  • Flatbed electrophoresis unit
  • 10 × 20– and 20 × 20–cm wicks (Schleicher & Schuell 903 paper; #35420)
  • 20 × 20 cm cellulose, glass‐backed TLC plate without fluorescence indicator (EM Science #5716 VWR)
  • Two 1/32‐in.‐thick, 20 × 20–cm Teflon sheets
  • 20 × 20–cm, ⅜‐in. Neoprene pad
  • 20 × 20–cm‐thick gel blotting paper (Schleicher & Schuell #31550)
  • Chromatography chamber (e.g., Fisher, VWR Scientific)
  • Whatman 3MM filter paper
  • Additional reagents and equipment for immunoblotting (unit 8.10) and autoradiography ( appendix 3A)
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Literature Cited

Literature Cited
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   Aebersold, R., Watts, J.D., Morrison, H.D., and Bures, E.J. 1991. Determination of the site of tyrosine phosphorylation at the low picomole level by automated solid‐phase sequence analysis. Anal. Biochem. 199:51‐60.
   Anderson, N.G., Maller, J.L., Tonks, N.K., and Sturgill, T.W. 1990. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature (Lond.) 343:651‐653.
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   Siegel, J.N., Klausner, R.D., Rapp, U.R., and Samelson, L.E. 1990. T cell antigen receptor engagement stimulates c‐raf phosphorylation and induces c‐raf‐associated kinase activity via a protein kinase C‐dependent pathway. J. Biol. Chem. 265:18472‐18480.
   Ullrich, A. and Schlessinger, J. 1990. Signal transduction by receptors with tyrosine kinase activity. Cell 61:203‐212.
   Wang, Y., Fiol, C.J., DePaoli‐Roach, A.A., Bell, A.W., Hermodson, M.A., and Roach, P.J. 1988. Identification of phosphorylation sites in peptides using a gas‐phase sequencer. Anal. Biochem. 174:537‐547.
Key Reference
   Boyle, W.J., van der Geer, P., and Hunter, T. 1991. Phosphopeptide mapping and phosphoamino acid analysis by two‐dimensional separation on thin‐layer cellulose plates. Methods Enzymol. 201:110‐149.
  Contains a detailed description of the underlying principles of phosphopeptide mapping and phosphoamino acid analysis and describes a theoretical basis for predicting migration of phosphopeptides, which is helpful in determining their identity.
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