Identifying Novel Protein‐Protein Interactions Using Co‐Immunoprecipitation and Mass Spectroscopy

R. Benjamin Free1, Lisa A. Hazelwood1, David R. Sibley1

1 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.28
DOI:  10.1002/0471142301.ns0528s46
Online Posting Date:  January, 2009
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Abstract

Proteomics has evolved from genomic science due to the convergence of advances in protein chemistry, separations, mass spectroscopy, and peptide and protein databases. Where identifying protein‐protein interactions was once limited to yeast two‐hybrid analyses or empirical data, protein‐protein interactions can now be examined in both cells and native tissues by precipitation of the protein complex of interest. Coupling this field to receptor pharmacology has recently allowed for the identification of proteins that differentially and selectively interact with receptors and are integral to their biological effects. It is becoming increasingly apparent that receptors in neurons do not exist as singular independent units, but rather are part of large macromolecular complexes of interacting proteins. It is a primary quest of neuroscience to piece together these interactions and to characterize the regulatory signalplexes of all proteins. This unit presents co‐immunoprecipitation‐coupled mass spectroscopy as one way of identifying signalplex partners. Curr. Protoc. Neurosci. 46:5.28.1‐5.28.14. © 2009 by John Wiley & Sons, Inc.

Keywords: proteomics; neuroreceptors; signalplex; receptor interactions

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Identification of Protein‐Protein Interactions Using an Affinity‐Tagged Bait Protein Expressed in Cultured Cells
  • Basic Protocol 2: Identification of Protein‐Protein Interactions Using Endogenous Bait Proteins in Brain Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identification of Protein‐Protein Interactions Using an Affinity‐Tagged Bait Protein Expressed in Cultured Cells

  Materials
  • Plasmid for expressing FLAG‐tagged protein of interest
  • Cultured cells of interest (cell line that originated from the endogenous tissue of interest, e.g., neurons for neuronal bait proteins, or cells capable of high levels of expression)
  • Cell culture medium
  • Transfection reagent/method (e.g., lipofectamine from Invitrogen, Neuroporter from Genelantis, or calcium phosphate from BD biosciences)
  • Earle's buffered salt solution (EBSS; or similar buffer suited for mammalian cell culture work) containing 5 mM EDTA
  • Solubilization buffer (see recipe), ice cold
  • Protease inhibitor cocktail (e.g., Complete Mini from Roche)
  • Protein‐A or ‐G agarose beads (or other conjugated agarose beads, matched for antibody class and species used in the IP; consult the protein agarose manufacturer for a list of species compatibility; alternatively, a blend of protein A/G agarose may be used)
  • Agarose beads coupled to anti‐FLAG antibody (e.g., Sigma anti‐M2 agarose)
  • Tris‐EDTA (TE) buffer, pH 7.4
  • Protein sample buffer (e.g., 2× LDS sample buffer from Invitrogen)
  • Reducing agent (e.g., sample reducing agent from Invitrogen)
  • Pre‐cast acrylamide gels (4% to 12% Bis‐Tris or similar)
  • Running and transfer buffer for acrylamide gels
  • Antibody directed against protein of interest (or against epitope tag)
  • G‐250 Coomassie stain (see recipe) or commercially available colloidal Coomassie stain (e.g., Simply Blue SafeStain from Invitrogen)
  • Coomassie de‐stain (see recipe)
  • Mass spectroscopy‐compatible silver stain (e.g., Silver Quest from Invitrogen), optional
  • 150‐mm tissue culture plates
  • Cell culture incubator
  • 10‐ml serological pipets
  • 50‐ml conical tubes
  • Refrigerated centrifuge
  • Orbital shaker, 4°C
  • 1.5‐ml screw‐top microcentrifuge tubes with rubber sealing O‐rings (e.g., screw‐top tubes from Axygen Scientific)
  • 37°C water bath
  • Gel running and blotting system (e.g., Novex mini‐cell and X‐Cell II blot module from Invitrogen)
  • PVDF membranes (e.g., Invitrolon from Invitrogen) or nitrocellulose membranes
  • Gel photographing equipment
  • Sterile razor blades
  • Additional reagents and equipment for PCR (Chapter 4), cell transfection ( appendix 11)
NOTE: It is critical that the samples be treated as sterile throughout the experimentation. MS analysis is extremely sensitive and any common contaminating proteins (i.e., keratin) can ruin protein detection. Always wear gloves, and use sterile sample tubes and filter‐tipped pipets and pipet tips.

Basic Protocol 2: Identification of Protein‐Protein Interactions Using Endogenous Bait Proteins in Brain Tissue

  Materials
  • Wild‐type mice
  • Knockout mice lacking protein of interest (optional)
  • Liquid N 2
  • Solubilization buffer (see recipe), ice cold
  • Protein‐A or ‐G agarose beads (or other conjugated agarose beads, matched for antibody class and species used in the IP; consult protein agarose manufacturer for a list of species compatibility; or alternately, use a blend of protein A/G agarose)
  • Non‐immune antibody (monocolonal control) or pre‐immune sera (polyclonal control) of same species and type
  • Antibody to protein of interest with demonstrated ability to immunoprecipitate
  • Mincing dishes or small beakers
  • Surgical scissors
  • 50‐ml centrifuge tubes
  • Polytron electric homogenizer with a small probe
  • 1.5‐ml screw‐top sterile centrifuge tubes with rubber sealing rings (e.g., Axygen Scientific)
  • Refrigerated microcentrifuge
  • Rocking platform, 4°C
  • Additional reagents and equipment for washing beads, eluting and separating protein, and preparing samples for mass spectrometry (see protocol 1)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
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Figures

Videos

Literature Cited

Literature Cited
   Becamel, C., Alonso, G., Galeotti, N., Demey, E., Jouin, P., Ullmer, C., Dumuis, A., Bockaert, J., and Marin, P. 2002. Synaptic multiprotein complexes associated with 5‐HT(2C) receptors: A proteomic approach. EMBO J. 10:2332‐2342.
   Free, R.B., Hazelwood, L.A., Cabrera, D.M., Spalding, H.N., Namkung, Y., Rankin, M.L., and Sibley, D.R. 2007. D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin. J. Biol. Chem. 282:21285‐21300.
   Husi, H., Ward, M., Choudhary, J., Blackstock, W., and Grant, S. 2000. Proteomic analysis of NMDA receptor‐adhesion protein signaling complexes. Nat. Neurosci. 3:661‐669.
   Kim, M., Jiang, L.H., Wilson, H.L., North, R., and Surprenant, A. 2001. Proteomic and functional evidence for a P2X7 receptor signalling complex. EMBO J. 22:6347‐6358.
   Sinz, A. 2003. Chemical cross‐linking and mass spectrometry for mapping three‐dimensional structures of proteins and protein complexes. J. Mass Spectrom. 38:1225‐1237.
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