Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry

Adam Brymora1, Valentina A. Valova1, Phillip J. Robinson1

1 Children's Medical Research Institute, Westmead NSW
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.5
DOI:  10.1002/0471143030.cb1705s22
Online Posting Date:  May, 2004
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Abstract

The aim of this unit is to provide a method for the identification of new protein‐protein interactions. Pull‐down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein‐protein interactions. When coupled with mass spectrometry, pull‐downs can be considered as the protein‐based equivalent of a yeast two‐hybrid screen. To improve the isolation of specific binding partners, pull‐down methods are described involving the use of cross‐linking, large‐scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state‐dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry‐based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in‐gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.

Keywords: Pull‐down; protein‐protein interactions; small GTPase; GST fusion protein; mass spectrometry; spin column; cross‐linking; in‐gel digestion; tissue lysate

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: The Pull‐Down Experiment
  • Alternate Protocol 1: Effector Isolation with GST‐Tagged Small Gtpases
  • Support Protocol 1: GST Fusion Protein Purification
  • Support Protocol 2: Cross‐Linking GST Fusion Proteins to GSH‐Agarose
  • Support Protocol 3: Preparation of Large‐Scale Tissue Lysates
  • Basic Protocol 2: Sample Preparation for Mass Spectrometry
  • Support Protocol 4: Recycling of GSH Agarose
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Pull‐Down Experiment

  Materials
  • Tissue lysate (see protocol 5)
  • Bead storage buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A)/50% (v/v) glycerol
  • GSH‐agarose beads (e.g., glutathione‐Sepharose 4B; Amersham Biosciences)
  • Wash buffers 1 or 2, and 3 (see reciperecipes)
  • 1× SDS sample buffer (unit 6.1)
  • Benchtop centrifuge, 4°C
  • Appropriately sized tubes that can be centrifuged
  • End‐over‐end rotator
  • Empty plastic column (e.g., Poly‐Prep from Bio‐Rad; to suit lysate volume) or Miracloth (Calbiochem)
  • MicroSpin columns (Amersham Biosciences), or other spin columns (see Table 17.5.1)
  • 85°C water bath
  • Additional reagents and equipment for preparing GST fusion proteins attached to GSH‐agarose beads (see protocol 3), cross‐linking of fusion proteins (optional; see protocol 4), preparing tissue lysate (see protocol 5), and SDS‐PAGE (unit 6.1)

Alternate Protocol 1: Effector Isolation with GST‐Tagged Small Gtpases

  • 1 M MgCl 2 ( appendix 2A)
  • Bead storage buffer (20 mM Tris·Cl, pH 7.4 ( appendix 2A)/50% (v/v) glycerol) containing 2.5 mM MgCl 2 (added from 1 M MgCl 2 stock; appendix 2A)
  • Small GTPase loading buffer (see recipe)
  • 100 mM GDP and GTP (or GTPγS) stock solutions (prepare fresh on the day of the experiment and keep cold):
    • 100 mM GDP (mol. wt. 463.2) = 1 mg in 21.6 µl H 2O
    • 100 mM GTP (mol. wt. 567.1) = 1 mg in 17.6 µl H 2O
    • 100 mM GTPγS (mol. wt. 627.2) = 1 mg in 15.9 µl H 2O
  • Wash buffers 4 or 5, and 6 (see reciperecipes)

Support Protocol 1: GST Fusion Protein Purification

  Materials
  • Glycerol stock of E. coli cells containing GST fusion protein expression vector (see appendix 3A and Coligan et al., )
  • LB medium and LB agar plates (see appendix 2A) containing 100 µg/ml ampicillin (or other appropriate antibiotic selection for GST expression vector used)
  • 100 mM isopropyl‐1‐thio‐β‐D‐galactoside (IPTG), filter sterilized
  • Bleach (e.g., Clorox)
  • Bacterial lysis buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A)/250 mM NaCl
  • 100 mg/ml lysozyme
  • 100 mM PMSF ( appendix 2A)
  • 20 mg/ml leupeptin
  • Liquid nitrogen and appropriate storage canister
  • 1 mg/ml DNase I
  • 10% (v/v) Triton X‐100 ( appendix 2A)
  • Bacterial lysis buffer (see above) containing 1% (v/v) Triton X‐100
  • 1% (w/v) SDS
  • Wash buffer 1 (see recipe)
  • Bead storage buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A)/50% (v/v) glycerol
  • 1× and 3× SDS sample buffer (unit 6.1)
  • Bovine serum albumin (BSA; optional)
  • Shaking bacterial incubator
  • Culture tubes, sterile
  • 1‐liter conical flasks (Pyrex or autoclavable plastic), sterilized by autoclaving or baking
  • Bugstoppers (Whatman), sterilized by autoclaving, or other sterile, gas‐permeable flask closures
  • Probe sonicator (e.g., Branson)
  • Refrigerated high‐speed centrifuge and benchtop centrifuge
  • 15‐ml and 50‐ml polypropylene screw‐cap tubes (e.g., Falcon)
  • Pipet tip cut to increase opening to >1 mm
  • 85°C water bath
  • MicroSpin columns (Amersham Biosciences)
  • Additional reagents and equipment for growing bacterial cells (see appendix 3A), SDS‐PAGE (unit 6.1), Coomassie blue staining of gels (unit 6.6)

Support Protocol 2: Cross‐Linking GST Fusion Proteins to GSH‐Agarose

  Materials
  • GST fusion protein beads (prepared as in protocol 3, with the protein concentration determined)
  • PBS ( appendix 2A)
  • Disuccinimidyl suberate (DSS), 13.2 mg in screw‐cap tube (see recipe)
  • bis(sulfosuccinimidyl) suberate (BS3; see recipe)
  • Dimethylsulfoxide (DMSO; H 2O‐free, i.e., fresh)
  • Quenching buffer: 1 M Tris·Cl, pH 8.2 ( appendix 2A), room temperature
  • Glutathione wash buffer (see recipe), room temperature
  • Wash buffer 1 (see recipe), ice‐cold
  • Cross‐linking wash buffer: 25 mM Tris·Cl, pH 7.6 ( appendix 2A)/1.5 M NaCl, ice‐cold
  • Bead storage buffer: 20 mM Tris·Cl, pH 7.4 ( appendix 2A)/50% (v/v) glycerol
  • Appropriately sized columns (e.g., Bio‐Rad 2‐ml Bio‐Spin columns or 10‐ml Poly‐Prep columns)

Support Protocol 3: Preparation of Large‐Scale Tissue Lysates

  Materials
  • 200 g sheep brain (see recipe)
  • Buffers A, B, C, and D (see reciperecipes; prepare in advance and cool overnight to 4°C)
  • GSH‐agarose beads (e.g., glutathione‐Sepharose 4B; Amersham Biosciences)
  • 10% (v/v) Triton X‐100
  • Colander or tea strainer
  • 1‐liter plastic beakers
  • Ultra‐Turrax tissue homogenizer (IKA Labortechnik) with 25‐mm‐diameter blade/tip
  • Refrigerated centrifuge and 0.5‐liter centrifuge bottles
  • Miracloth (Calbiochem)
  • 50‐ml screw‐cap plastic tubes
NOTE: Sometimes precipitates accumulate during freeze/thaw of these samples, and this is particularly true for the peripheral membrane extract (see note above). All frozen aliquots must be centrifuged after defrosting at 35,000 × g (or maximum speed) for 30 min at 4°C, before incubation with recombinant GST fusion protein beads. It is essential to clear any debris before adding the extract to the beads. It may also be useful to pre‐clear the cytosol extracts a third time before use, to remove any remaining traces of endogenous GST (this is optional; see protocol 1 or the protocol 2Alternate Protocol). All volumes of beads in this protocol refer to bed volumes.

Basic Protocol 2: Sample Preparation for Mass Spectrometry

  Materials
  • Unstained SDS acrylamide gel ( protocol 1 or protocol 2Alternate Protocol)
  • Colloidal Coomassie stain (see recipe)
  • 1% (v/v) acetic acid
  • Zinc stain solutions I and II (see reciperecipes)
  • 50% (v/v) and 100% acetonitrile
  • 25 and 50 mM ammonium bicarbonate (prepare from 1 M stock; see recipe)
  • 25 mM ammonium bicarbonate in 50% (v/v) acetonitrile (prepare from 1 M ammonium bicarbonate stock; see recipe)
  • Zinc destain solution (see recipe)
  • Digestion buffer (see recipe), ice‐cold
  • 5% (v/v) formic acid
  • 50% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA)
  • Sealable plastic containers of appropriate size for gel
  • Platform shaker
  • Flatbed scanner (preferably with transparency adapter for Coomassie stain)
  • Computer running graphics software, e.g., Adobe Photoshop
  • Light box
  • Scalpel (with new blades) or fine‐gauge needles
  • Siliconized, hydrophobically coated, or highly polished plastic microcentrifuge tubes (e.g., Axygen Maxymum recovery clear 0.6‐ml tubes), pipet tips (Axygen Maxymum recovery pipet tips), and gel‐loading tips (e.g., Sorenson Multi Miniflex round sterile gel loading pipet tips, 0.1 to 10 µl)
  • Stainless steel mortar and pestle (e.g., Quantum Scientific)
  • Heat‐sealable plastic sleeves and heat sealer
  • Pieces of cardboard and large sturdy flat box to accommodate gels
  • 50‐ml screw‐cap tubes
  • Heavy‐gauge needle
  • Thermomixer/shaker (to fit microcentrifuge tubes)
  • Bath sonicator

Support Protocol 4: Recycling of GSH Agarose

  Materials
  • Beads for recycling (from pre‐clearing steps in protocol 1, the protocol 2Alternate Protocol, or protocol 5)
  • Glutathione wash buffer (see recipe), room temperature
  • Bead recycling buffer: 100 mM glycine hydrochloride, pH 2.3, room temperature
  • Phosphate buffered saline (PBS; appendix 2A), room temperature
  • 20% (v/v) ethanol
  • 1% (w/v) SDS (see appendix 2A)
  • 0.1% (v/v) Triton X‐100 (see appendix 2A)
  • End‐over‐end rotator
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Figures

Videos

Literature Cited

Literature Cited
   Berggren, K.N., Schulenberg, B., Lopez, M.F., Steinberg, T.H., Bogdanova, A., Smejkal, G., Wang, A., and Patton, W.F. 2002. An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation. Proteomics 2:486‐498.
   Brymora, A., Cousin, M.A., Roufogalis, B.D., and Robinson, P.J. 2001a. Enhanced protein recovery and reproducibility from pull‐down assays and immunoprecipitations using spin columns. Anal. Biochem. 295:119‐122.
   Brymora, A., Valova, V.A., Larsen, M.R., Roufogalis, B.D., and Robinson, P.J. 2001b. The brain exocyst complex interacts with RalA in a GTP‐dependent manner: Identification of a novel mammalian Sec3 gene and a second Sec15 gene. J. Biol. Chem. 276:29792‐29797.
   Cantor, S.B., Urano, T., and Feig, L.A. 1995. Identification and characterization of Ral‐binding protein 1, a potential downstream target of Ral GTPases. Mol. Cell. Biol. 15:4578‐4584.
   Castellanos‐Serra, L. and Hardy, E. 2001. Detection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: A decade of research. Electrophoresis 22:864‐873.
   Coligan, J.E., Dunn, B.M., Speicher, D.W., and Wingfield, P.T. (eds.). 2003. Current Protocols in Protein Science. John Wiley & Sons, New York.
   Fernandez‐Patron, C., Hardy, E., Sosa, A., Seoane, J., and Castellanos, L. 1995. Double staining of Coomassie blue‐stained polyacrylamide gels by imidazole‐sodium dodecyl sulfate‐zinc reverse staining: Sensitive detection of Coomassie blue–undetected proteins. Anal. Biochem. 224:263‐269.
   Frech, M., Schlichting, I., Wittinghofer, A., and Chardin, P. 1990. Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli. J. Biol. Chem. 265:6353‐6359.
   Glebska, J., Grzelak, A., Pulaski, L., and Bartosz, G. 2002. EDTA loses its antioxidant properties upon storage in buffer. Anal. Biochem. 311:87‐89.
   Graves, P.R. and Haystead, T.A. 2002. Molecular biologist's guide to proteomics. Microbiol. Mol. Biol. Rev. 66:39‐63.
   Harper, S. and Speicher, D.W. 1997. Expression and purification of GST fusion proteins. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.) pp. 6.6.1‐6.6.21. John Wiley & Sons, New York.
   Larsen, M.R. and Roepstorff, P. 2000. Mass spectrometric identification of proteins and characterization of their post‐translational modifications in proteome analysis. Fresenius J. Anal. Chem. 366:677‐690.
   Menard, L., Tomhave, E., Casey, P.J., Uhing, R.J., Snyderman, R., and Didsbury, J.R. 1992. Rac1, a low‐molecular‐mass GTP‐binding‐protein with high intrinsic GTPase activity and distinct biochemical properties. Eur. J. Biochem. 206:537‐546.
   Moskalenko, S., Henry, D.O., Rosse, C., Mirey, G., Camonis, J.H., and White, M.A. 2002. The exocyst is a Ral effector complex. Nat. Cell Biol. 4:66‐72.
   Moskalenko, S., Tong, C., Rosse, C., Camonis, J., and White, M.A. 2003. Ral GTPases regulate exocyst assembly through dual subunit interactions. J. Biol. Chem. 278:51743‐51748.
   Neuhoff, V., Arold, N., Taube, D., and Ehrhardt, W. 1988. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250. Electrophoresis 9:255‐262.
   Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. 1996. Mass spectrometric sequencing of proteins silver‐stained polyacrylamide gels. Anal. Chem. 68:850‐858.
   Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K., and Ohta, Y. 2002. The exocyst complex binds the small GTPase RalA to mediate filopodia formation. Nat. Cell Biol. 4:73‐78.
   Takai, Y., Sasaki, T., and Matozaki, T. 2001. Small GTP‐binding proteins. Physiol. Rev. 81:153‐208.
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