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

  •   FigureFigure 13.9.1 Sample and dye origins and blotter dimensions for separation of peptides at different pH values. (A) Location of the sample and dye origins for electrophoresis at pH 1.9 and pH 4.72 and (B) at pH 8.9. To mark a TLC plate, the plate is placed on top of a life‐sized template. This is then placed on top of a light box and the origins are marked on the cellulose side using a very blunt extra‐soft pencil. Dimensions of the blotter and the location of two holes that fit over the sample and dye origins are shown (C) for electrophoresis at pH 1.9 and 4.72 and (D) for pH 8.9.
  •   FigureFigure 13.9.2 Separation of peptides by electrophoresis.(A) The sample and dye are spotted on their respective origin at the bottom and the top of the TLC plate as described in the text. (B) The blotter is soaked briefly in the electrophoresis buffer, and excess liquid is removed by blotting briefly on a piece of 3MM filter paper. (C) The TLC plate is wetted by placing the wetted blotter on top, with the sample and marker origins in the centers of the two holes. The blotter is pressed onto the TLC plate around the sample and marker origins to ensure uniform flow of electrophoresis buffer from the blotter towards the sample and marker origins. This will result in concentration of the sample and marker dye on their respective origins, and will improve resolution. The rest of the blotter is pressed with a flat hand onto the TLC plate, the blotter is removed, and the plate is examined; it should be dull gray with no shiny puddles of buffer. Excess buffer should be allowed to evaporate or be blotted very carefully with tissue paper. The plate is placed on the apparatus and the electrophoresis run for 20 to 30 min at 1 kV. This results in separation of the peptides in the first dimension (D, peptides shown in black). The position of the anode and cathode are indicated in panel D.
  •   FigureFigure 13.9.3 Preparation of the HTLE 7000 electrophoresis system.
  •   FigureFigure 13.9.4 Separation of peptides in the second dimension by chromatography. After electrophoresis, air‐dry the plate. A fan may be used to facilitate this. Add a small amount of green marker dye in the left hand (A) or right hand margin at the same level as the sample origin. Place the plate(s) almost upright in a chromatography tank, replace the lid, and run the chromatography until the buffer front reaches to within 1 to 2 cm from the top of the TLC plate (B and C). This results in separation of the peptides in the second dimension (D; peptides shown in black). Open the tank, take out all plates, let the plates air dry, apply fluorescent ink at the margins of the plate, and expose to X‐ray film.
  •   FigureFigure 13.9.5 Sample and dye origins and blotter dimensions for analysis of manual Edman degradation products at pH 1.9. (A) Location of the sample and standard origins. To mark a TLC plate, the plate is placed on top of a life‐sized template on top of a light box and the origins are marked on the cellulose side using a very blunt extra‐soft pencil. (B) Dimensions of the blotter and the location of the slot that fits over multiple sample and marker origins. The blotter is soaked in electrophoresis buffer, blotted with a sheet of Whatman paper to remove most of the buffer, and placed on top of the TLC plate so that the origins are in the middle of the slot.
  •   FigureFigure 13.9.6 An example of a tryptic phosphopeptide map based on that of human Nck‐alpha. For the first (horizontal) dimension, electrophoresis was run at pH 1.9 for 25 min at 1.0 kV; the anode is at the left. Ascending chromatography was run for 15 hr in phosphochromo buffer. The sites represented by spots 1 to 7, with the exception of spot 2, have been identified. Spot 1 is an 11‐amino‐acid, phosphotyrosine‐containing peptide. While spot 2 also contains phosphotyrosine, and runs in a position likely to be the doubly phosphorylated version of this peptide, it turns out to be unrelated to spot 1. Spot 3 represents a 5‐amino‐acid, phosphoserine‐containing peptide; this same peptide with an amino‐terminal arginine runs as spot 4; thus it can be seen that in this case the tryptic cleavage is largely incomplete. Spot 5 represents a 20‐amino‐acid phosphoserine‐containing peptide which, with an amino‐terminal lysine, runs as spot 6. Spot 7 represents a peptide that is unrelated to 5 and 6. Spot 8 represents free phosphate, released during sample preparation.

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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