Identification of Protein‐Protein Interactions by Surface Plasmon Resonance followed by Mass Spectrometry

Alexandra Madeira1, Elisabet Vikeved1, Anna Nilsson2, Benita Sjögren1, Per E. Andrén2, Per Svenningsson1

1 Karolinska Institute, Stockholm, Sweden, 2 Uppsala University, Biomedical Centre, Uppsala, Sweden
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.21
DOI:  10.1002/0471140864.ps1921s65
Online Posting Date:  August, 2011
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Elucidation of the function and meaning of the protein networks can be useful in the understanding of many pathological processes and the identification of new therapeutic targets. This unit describes an approach to discover protein‐protein interactions by coupling surface plasmon resonance to mass spectrometry. Briefly, a protein is covalently bound to a sensor chip, which is then exposed to brain extracts injected over the surface via a microfluidic system. This allows the monitoring in real‐time of the interactions between the immobilized ligand and the extracts. Interacting proteins from the extracts are then recovered, trypsinized, and identified using mass spectrometry. The data obtained are searched against a sequence database using the Mascot software. To exclude nonspecific interactors, control experiments using blank sensor chips, and/or randomized peptides, are performed. The protocol presented here does not require specific labeling or modification of proteins and can be performed in <4 days. Curr. Protoc. Protein Sci. 65:19.21.1‐19.21.9. © 2011 by John Wiley & Sons, Inc.

Keywords: SPR; mass spectrometry; protein interactions

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • HBS‐EP buffer, pH 7.4 (Biacore Life Sciences)
  • Protein used as ligand, diluted in immobilization buffer (100 µg/ml; final volume: 500 µl); prepare immediately before use
  • Immobilization buffer (see recipe)
  • Amine Coupling kit (Biocore Life Sciences) containing:
    • 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC)
    • N‐hydroxysuccinimide (NHS)
    • 1.0 M ethanolamine
  • 1% (v/v) Acetic acid
  • HBS‐N buffer, pH 7.4 (Biacore Life Sciences)
  • 10 mM Octyl β‐D‐glucopyranoside (OGP; Sigma) in HBS‐N buffer, pH 7.4 (Biacore Life Sciences)
  • Brain extracts (see Ohman et al., )
  • 50 mM OGP in brain extracts protein solution [50 µg/ml in Tris‐buffered saline (TBS; see recipe)], prepare immediately before use
  • 50 mM NaOH
  • 50 mM OGP in 2% (v/v) acetic acid, prepare immediately before use
  • Recovery solution (see recipe)
  • Deionized water
  • Trypsin solution (see recipe)
  • 50 mM ammonium bicarbonate (NH 4HCO 3)
  • 0.25% (v/v) acetic acid
  • Solution B (see recipe)
  • Biacore 3000 SPR Sensor with a control software version 4.0 (Biacore Life Sciences)
  • Sensor Chip CM5 (research grade; Biacore Life Sciences)
  • 7‐mm plastic vials (Biacore Life Sciences)
  • 16‐mm glass vials (Biacore Life Sciences)
  • LoBind tubes (Eppendorf)
  • Eppendorf SpeedVac Concentrator 5301 (Eppendorf)
  • Polypropylene insert, 100 µl (Agilent)
  • Ettan Multidimensional Liquid Chromatography (MDLC) system (GE Healthcare)
  • Glass vials with blue screw caps (Agilent)
  • Unicorn 5.01 (GE Healthcare)
  • LTQ Linear Ion Trap (Thermo Scientific)
  • High‐pressure Column Packer and Sample Loader operated at 50 to 60bars (Proxeon Biosystems)
  • C18 PepMap100 solid‐phase extraction m‐precolumn cartridge (particle size 5 mm, pore size 100 Å, 300‐mm inner diameter; Dionex)
  • Fused silica emitter tip (375‐µm o.d., 75‐µm i.d., 6‐µm tip, 150‐mm length; Proxeon Biosystems)
  • Xcalibur 1.4 SR1 software (Thermo Scientific)
  • Software tool for compiling dta files into mgf (
  • Mascot software (Matrix Science)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals (for preparation of 50 mM OGP in Brain extracts protein solution).NOTE: Degas and filter all reagents with a 0.22‐µm filter. This prevents the clogging of the microfluidic system.
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Literature Cited

Literature Cited
   Borch, J. and Roepstorff, P. 2004. Screening for enzyme inhibitors by surface plasmon resonance combined with mass spectrometry. Anal. Chem. 76:5243‐5248.
   Buijs, J. and Franklin, G.C. 2005. SPR‐MS in functional proteomics. Brief. Funct. Genomics Proteomics 4:39‐47.
   Chatelier, R.C., Gengenbach, T.R., Griesser, H.J., Brigham‐Burke, M., and O'Shannessy, D.J. 1995. A general method to recondition and reuse BIAcore sensor chips fouled with covalently immobilized protein/peptide. Anal. Biochem. 229:112‐118.
   Krone, J.R., Nelson, R.W., Dogruel, D., Williams, P., and Granzow, R. 1997. BIA/MS: Interfacing biomolecular interaction analysis with mass spectrometry. Anal. Biochem. 244:124‐132.
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   Madeira, A., Ohman, E, Nilsson, A, Sjögren, B, Andrén, P.E., and Svenningsson, P. 2009. Coupling surface Plasmon resonance to mass spectrometry to discover novel protein‐protein interactions. Nature Protoc. 4:1023‐1037.
   Madeira, A., Yang, J., Zhang, X., Vikeved, E., Nilsson, A., Andrén, P.E., and Svenningsson, P. 2011. Caveolin‐1 interacts with alpha‐synuclein and mediates toxic actions of cellular alpha‐synuclein overexpression. Neurochem. Int. 2011 June 13. [Epub ahead of print]
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   Ohman, E., Nilsson, A., Madeira, A., Sjogren, B., Andren, P.E., and Svenningsson, P. 2008. Use of surface plasmon resonance coupled with mass spectrometry reveals an interaction between the voltage‐gated sodium channel type X alpha‐subunit and caveolin‐1. J. Proteome Res. 7:5333‐5338.
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