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

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

¹National 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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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 1)

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 N₂
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 Basic 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

Figure 5.28.1

Overview of procedure for identifying novel protein-protein interactions using co-immunoprecipitation and mass spectroscopy.

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Figure 5.28.2

Identification of D₁ receptor–interacting proteins using immunoprecipitation and mass spectroscopy. (A) Coomassie-blue stained gel of immunoprecipitated proteins. The D₁-FLAG lane shows proteins immunoprecipitated from cells expressing the FLAG-D₁ receptor. The FLAG-tag lane shows proteins immunoprecipitated from cells only expressing the FLAG peptide. Band number indicates the band cut from the gel and subjected to MS-based sequencing. Band 1 was found to be the interacting protein calnexin. Band 2 was found to be the parent bait protein, the D₁ dopamine receptor. Neither of these proteins was found in the control lane. (B) Representative MS/MS spectrum obtained after excision of band 1 and fragmentation of the precursor ion at m/z 886.6. LC-MS/MS (tandem mass spectrometry) was carried out on the peptide mixture obtained from in-gel digestion of SDS-PAGE-separated protein samples with LCQ ion trap mass spectrometer on-line coupled with an HPLC with 75-µm i.d. C18 column. The precursor ions were selected automatically by the instrument. The peptide sequence was identified by bioinformatics analysis and found to correspond to the parent protein calnexin. This figure is adapted from Free et al. (2007), with permission.

View Image
Figure 5.28.3

Verification of interaction via immunoblot analysis. Left panel: HEK293T cells were transfected with D₁-FLAG or vector containing only the FLAG peptide (FLAG-tag). Proteins were extracted, immunoprecipitated (IP) using anti-FLAG agarose, separated by SDS-PAGE and immunoblotted (IB). Blots were probed with a monoclonal anti-D₁ antibody and visualized using enhanced chemiluminescence (ECL) after incubation with an anti-rat HRP-conjugated antibody. Right panel: The blot in the left panel was stripped of all antibodies, re-probed with an anti-calnexin antibody and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. These data confirm the mass spectroscopy findings in Figure 5.28.2 and support an interaction between the proteins. This figure is adapted from Free et al. (2007), with permission.

View Image

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.