Phosphopeptide Mapping and Identification of Phosphorylation Sites

Jill Meisenhelder1, Tony Hunter1, Peter van der Geer2

1 The Salk Institute for Biological Studies, La Jolla, 2 University of California, San Diego, La Jolla
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.9
DOI:  10.1002/0471140864.ps1309s18
Online Posting Date:  May, 2001
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Abstract

Protein phosphorylation is a common modification for many proteins in the cell. Phosphorylation can affect localization of a protein, its stability, and its ability to dimerize or form stable complexes with other molecules. To understand the underlying mechanisms behind the phosphorylation of a given protein, it is often necessary to precisely identify which amino acid residues are phosphorylated. This unit describes the technique of phosphopeptide mapping. In this procedure, a radiolabeled protein is proteolytically digested, and the resulting phosphopeptides are separated in two dimensions on a TLC plate. The phosphopeptides are also analyzed by HPLC and mass spectrometry or peptide microsequencing. Such mapping gives information about the number of phosphorylation sites present in the protein, and can also be used to find out if sites of phosphorylation on a protein change upon treatment of cells with specific agents.

     
 
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Table of Contents

  • Basic Protocol 1: Tryptic Phosphopeptide Mapping of Proteins Isolated from SDS‐Polyacrylamide Gels
  • Alternate Protocol 1: Proteolytic Digestion of Immobilized Proteins
  • Support Protocol 1: Isolation of Phosphopeptides from the Cellulose Plate
  • Basic Protocol 2: Determination of the Position of the Phosphorylated Amino Acid in the Peptide by Manual Edman Degradation
  • Basic Protocol 3: Diagnostic Secondary Digests to Test for the Presence of Specific Amino Acids in the Phosphopeptide
  • Support Protocol 2: Preparation of Phosphopeptides for Microsequence Determination or Mass Spectrometry
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Tryptic Phosphopeptide Mapping of Proteins Isolated from SDS‐Polyacrylamide Gels

  Materials
  • Samples containing 32P‐labeled proteins of interest (unit 13.2)
  • Fluorescent ink or paint (can be obtained from most arts and crafts supply stores)
  • 50 mM ammonium bicarbonate, pH 7.3 to 7.6 (when freshly prepared the buffer has a pH of ∼7.5), and pH 8.0 (the pH drifts overnight to ∼8.0, ideal for digestion with trypsin or chymotrypsin as in step )
  • 2‐Mercaptoethanol (2‐ME)
  • 20% (w/v) SDS ( appendix 2E)
  • 50 mM ammonium bicarbonate, pH 7.3 to 7.6, containing 0.1% (w/v) SDS and 1.0% (v/v) 2‐ME
  • 2 mg/ml carrier protein (RNase A, BSA, or immunoglobulins) in deionized water (store in aliquots at −20°C or −70°C)
  • 100% (w/v) trichloroacetic acid (TCA)
  • 96% ethanol, ice cold
  • 30% (w/v) hydrogen peroxide
  • 98% (w/v) formic acid
  • 1 mg/ml TPCK‐treated trypsin (e.g., Worthington) in deionized water or 0.1 mM HCl (store in aliquots at −70°C or under liquid nitrogen)
  • recipeElectrophoresis buffers (see recipe): pH 1.9, 3.5, 4.72, 6.5, and 8.9
  • recipeGreen marker dye (see recipe)
  • recipeChromatography buffer (see recipe; also see Critical Parameters for discussion of buffer selection)
  • Single‐edge razor blades or surgical blades
  • Scintillation counter appropriate for Cerenkov counting
  • 1.7‐ml screw‐cap microcentrifuge tubes (Sarstedt)
  • Disposable tissue‐grinder pestles (Kontes)
  • Platform rocker
  • Tabletop centrifuge with swinging‐bucket rotor
  • 1.5 ml microcentrifuge tube, pretested for suitability (see Critical Parameters)
  • Glass‐backed TLC plates (20 × 20 cm, 100 µm cellulose; EM Science)
  • Low‐volume adjustable pipet with long disposable tips made of flexible plastic, e.g., gel‐loading tips
  • Air line fitted with filter to trap aerosols and particulate matter
  • HTLE 7000 electrophoresis apparatus (CBS Scientific)
  • Polyethylene sheeting (35 × 25 cm; CBS Scientific)
  • Electrophoresis wicks (20 × 28 cm sheet of Whatman 3MM paper folded lengthwise to give double thickness sheets of 20 × 14 cm)
  • Chromatography tank (CBS Scientific)
  • Fan for drying TLC plates
  • 65°C drying oven
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and autoradiography (unit 10.11)

Alternate Protocol 1: Proteolytic Digestion of Immobilized Proteins

  • Methanol
  • recipe0.5% (w/v) PVP‐360 in 100 mM acetic acid (see recipe)
  • 50 mM ammonium bicarbonate, pH 8.0
  • PVDF membrane (Immobilon P, Millipore) or nitrocellulose membrane (unit 10.10)
  • Saran Wrap or Mylar
  • Additional reagents and equipment for wet or semidry protein transfer (unit 10.10)

Support Protocol 1: Isolation of Phosphopeptides from the Cellulose Plate

  Materials
  • TLC plate with resolved phosphopeptides and corresponding autoradiogram (see protocol 1 or protocol 2)
  • recipeElectrophoresis buffer, pH 1.9 (see recipe)
  • Single‐edge razor blades
  • 1000‐µl (blue) pipet tips
  • Small sintered polyethylene disk to fit inside blue tips (Kontes)
  • Glass rod or similar instrument to push filters into tips

Basic Protocol 2: Determination of the Position of the Phosphorylated Amino Acid in the Peptide by Manual Edman Degradation

  Materials
  • Eluted phosphopeptide (see protocol 3)
  • 5% (v/v) phenylisothiocyanate (PITC) in pyridine
  • 10:1 (v/v) heptane/ethyl acetate—mix 10 parts heptane with 1 part ethyl acetate
  • 2:1 (v/v) heptane/ethyl acetate—mix 2 parts heptane with 1 part ethyl acetate
  • 100% (w/v) trifluoroacetic acid (TFA)
  • recipeElectrophoresis buffer, pH 1.9 (see recipe)
  • 200 to 500 cpm 32P (prepared by diluting 32P orthophosphate with deionized water) or recipe2 mg/ml PTH‐phosphotyrosine (see recipe)
  • 1.5‐ml microcentrifuge tubes, pretested for suitability (see Critical Parameters)
  • 45°C water bath
  • Scintillation counter appropriate for Cerenkov counting
  • Glass‐backed TLC plates (20 × 20 cm, 100 µm cellulose; EM Science)
  • 65°C drying oven or fan
  • Additional reagents and equipment for electrophoresis of peptides on a TLC plate (see protocol 1 and Figure ) and autoradiography (unit 10.11)

Basic Protocol 3: Diagnostic Secondary Digests to Test for the Presence of Specific Amino Acids in the Phosphopeptide

  Materials
  • Eluted phosphopeptide (see protocol 3)
  • Enzyme to be used for digestion and appropriate buffer (see Table 13.9.1)
    Table 3.9.1   MaterialsSpecificities and Digestion Conditions for Enzymes and Other Cleavage Reagents

    Enzyme or reagent Specificity a Digestion conditions Comments
    TPCK‐trypsin K–X;R–X pH 8.0‐8.3 Does not cut K/R‐P; cuts inefficiently at K/R‐X‐P.Ser/P.Thr and K/R‐D/E; cuts wells at K/R‐P.Ser/P.Thr; cuts X‐K/R‐K/R‐K/R incompletely
    α‐Chymotrypsin F–X;W–X; Y–X pH 8.3 Does not cleave F/W/Y‐P or P.Tyr‐X
    Thermolysin X–L; X–I; X–V pH 8.0, 1 mM CaCl 2, 55°C Will recognize most apolar residues to some extent; CaCl 2 may affect the electrophoretic mobility
    Proline‐specific endopeptidase P–X pH 7.6
    Cyanogen bromide (CNBr) M–X 50 mg/ml CNBr in 70% formic acid, 90 min, 23°C Toxic; will cleave only unoxidized methionine
    V8 protease E–X pH 7.6 Will not cleave at every E in whole proteins; does give a consistent pattern
    Endoproteinase Asp‐N X–CSO 3H;X–D pH 7.6 Will cleave X‐E at high enzyme concentrations
    Formic acid D–P 70% formic acid, 37°C, 24‐48 hr

     aThe dash indicates the cleavage site. See appendix 1A for definitions of the one‐letter abbreviations for amino acids.
  • 2‐Mercaptoethanol (2‐ME)
  • recipeElectrophoresis buffer of appropriate pH (see recipe)
  • Water bath at appropriate temperature for enzyme digestion
  • Glass‐backed TLC plates (20 × 20 cm, 100 µm cellulose; EM Science)
  • Additional reagents and equipment for chromatography and electrophoresis of phosphopeptides (see protocol 1, steps to )
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Figures

Videos

Literature Cited

Literature Cited
   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. 201:110‐148.
   Fischer, W.H., Karr, D., Jackson, B., Park, M., and Vale, W. 1991. Microsequence analysis of proteins purified by gel electrophoresis. Methods Neurosci. 6:69‐84.
   Fischer, W.H., Hoeger, C.A., Meisenhelder, J. Hunter, T., and Craig, A.G. 1997. Determination of phosphorylation sites in peptides and proteins employing a volatile Edman reagent. J. Protein Chem. 16:329‐333.
   Mitchelhill, K.I. and Kemp, B.E. 1999. Phosphorylation site analysis by mass spectrometry. In Protein Phosphorylation: A Practical Approach (D.G., ed.) pp. 127‐151. Oxford University Press, Oxford.
   Mitchelhill, K.I., Michell, B.J., House, C.M., Stapleton, D., Dyck, J., Gamble, J., Ullrich, C., Witters, L.A., and Kemp, B.E. 1997. Posttranslational modifications of the 5′‐AMP‐activated protein kinase β1 subunit. J. Biol. Chem. 272:24475‐24479.
   van der Geer, P. and Hunter, T. 1990. Identification of tyrosine 706 in the kinase insert as the major colony‐stimulated factor 1 (CSF‐1)–stimulated autophosphorylation site in the CSF‐1 receptor in a murine macrophage cell line. Mol. Cell. Biol. 10:2991‐3002.
   Wang, Y.K., Liao, P.‐C., Allison, J., Gage, D.A., Andrews, P.C., Lubman, D.M., Hanash, S.M., and Strahler, J.R. 1993. Phorbol 12‐myristate 13‐acetate‐induced phosphorylation of op18 in Jurkat T cells. J. Biol. Chem. 268:14269‐14277.
Key References
   Boyle et al., 1991. See above.
  Both of these papers discuss many of the protocols described in this unit.
   van der Geer, P., Luo, K. Sefton, B.M., and Hunter, T. 1993. Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin‐layer plates. In Protein Phosphorylation; a Practical Approach (Hardie, D.G., ed.) pp. 97‐126. IRL Press, Oxford.
Internet Resources
   http://www.genestream.org
  This Web site contains a program for calculating the mobility of a peptides of known composition and a program that reads the position of a spot on the actual map and calculates which peptide(s) derived from the protein being mapped could have the mobility of that spot.
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