Application of Amide Proton Exchange Mass Spectrometry for the Study of Protein‐Protein Interactions

Jeffrey G. Mandell1, Abel Baerga‐Ortiz1, Carrie H. Croy1, Arnold M. Falick1, Elizabeth A. Komives1

1 University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.9
DOI:  10.1002/0471140864.ps2009s40
Online Posting Date:  June, 2005
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Abstract

This protocol describes amide proton exchange experiments that probe for changes in solvent accessibility at protein‐protein interfaces. The simplest version of the protocol, termed the “on‐exchange” experiment, detects protein‐protein interfaces by taking advantage of the fact that solvent deuterium oxide (D2O) molecules are excluded from the surface of a protein to which another protein is bound. A more complete version of the experiment can also be performed in which the rate of surface deuteration is initially measured separately for each of the proteins involved in the interaction, after which the deuterated proteins are allowed to complex and the rate of “off‐exchange” (i.e., replacement of surface deuterons by protons from solvent H2O molecules) at the resulting protein‐protein interface is measured. This version of the experiment yields additional kinetic information that can help to define the solvent‐inaccessible “core” of the interface.

Keywords: antibody; epitope; hydration; thrombin

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Identification of the Protein‐Protein Interface Via the On‐Exchange Method
  • Support Protocol 1: Analysis of Experimental Data from the On‐Exchange Experiment
  • Basic Protocol 2: Determination of the Solvent Accessibility of a Protein‐Protein Interface Via the Off‐Exchange Method
  • Alternate Protocol 1: Determination of the Solvent Accessibility of a Protein‐Protein Interface Via the Off‐Exchange Method Using an Agarose‐Bound Interaction Partner
  • Support Protocol 2: Analysis of Off‐Exchange Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identification of the Protein‐Protein Interface Via the On‐Exchange Method

  Materials
  • 10× low‐salt buffer (e.g., 25 mM Tris·Cl, 25 mM NaCl, 50 mM Na/KHPO 4)
  • Purified receptor and ligand proteins
  • 0.1% and 2.0% (v/v) trifluoroacetic acid (TFA; highest purity possible)
  • 2 to 3 mg/ml immobilized pepsin in 6% (w/v) cross‐linked beaded agarose (Pierce Biotechnology)
  • Matrix solution for MALDI‐TOF mass spectrometry (see recipe)
  • Peptide mass standard mixture (e.g., Sequazyme Calibration Mixture 2; Applied Biosystems)
  • Deuterium oxide (D 2O; 99.996% purity; Cambridge Isotope Laboratories)
  • pH meter with InLab 423 electrode (Mettler Instruments)
  • Cold box, 4°C (Isotemp Chromatography Refrigerator; Fisher Scientific)
  • Microcentrifuge, 4°C (in cold box)
  • 0.5‐ml thin‐walled microcentrifuge tubes (USA/Scientific)
  • MALDI target plates, 4°C (in cold box; chilled overnight in a plastic case to prevent absorption of atmospheric H 2O)
  • Vacuum desiccator (with liquid nitrogen trap) located adjacent to MALDI‐TOF mass spectrometer
  • MALDI‐TOF mass spectrometer (unit 16.2, unit 16.3, and unit 16.4)
  • C 18 analytical column
  • Additional reagents and equipment for reversed‐phase high‐performance liquid chromatography (HPLC; unit 8.7) and post‐source decay sequencing (unit 16.1), carboxypeptidase Y C‐terminal sequencing (unit 11.8), or electrospray ionization tandem mass spectrometric (ESI MS/MS) sequencing (unit 16.10 and unit 16.11)
NOTE: The amide proton exchange reaction is highly sensitive to temperature; exchange is rapid at room temperature but slows dramatically when the reaction sample is chilled to 4°C. Thus, all sample manipulations subsequent to the timed incubation of the receptor, ligand, or receptor‐ligand complex in D 2O must be performed at 4°C. The simplest experimental setup that makes this possible involves having a cold box in the same room as the mass spectrometer.

Support Protocol 1: Analysis of Experimental Data from the On‐Exchange Experiment

  Materials
  • MALDI‐TOF mass spectra of pepsin digest products from all receptor, ligand, and receptor‐ligand complex on‐exchange samples ( protocol 1)
  • MALDI‐TOF mass spectra of pepsin digest products from nondeuterated receptor, ligand, and receptor‐ligand complex samples ( protocol 1)
  • Software for displaying multiple mass spectra in stacked layout (provided by manufacturer of the mass spectrometer)
  • CAPP software package (Mandell et al., 1988b)
  • KaleidaGraph 3.0 (Synergy Software) or other nonlinear fitting software

Basic Protocol 2: Determination of the Solvent Accessibility of a Protein‐Protein Interface Via the Off‐Exchange Method

  • H 2O buffer: 10 mM Tris·Cl ( appendix 2E), pH 6.6 to 7.9, as appropriate

Alternate Protocol 1: Determination of the Solvent Accessibility of a Protein‐Protein Interface Via the Off‐Exchange Method Using an Agarose‐Bound Interaction Partner

  • Antibody of interest
  • Protein G‐agarose beads (Sigma)
  • 20 mM dimethylpimelimidate (Pierce Chemical)
  • Epitope‐containing protein of interest
  • 1:1 mixture of 1‐propanol/0.1% (v/v) trifluoroacetic acid (TFA; pH 2.25), chilled to 4°C
  • Additional reagents and equipment for buffer exchange (unit 4.4)
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Figures

Videos

Literature Cited

Literature Cited
   Anand, G.S., Hughes, C.A., Jones, J.M., Taylor, S.S., and Komives, E.A. 2002. Amide H/2H exchange reveals communication between the cAMP‐ and catalytic subunit‐binding sites in the regulatory subunit of protein kinase A. J. Mol. Biol. 323:377‐386.
   Anand, G.S., Law, D., Mandell, J.G., Snead, A.N., Tsigelny, I., Taylor, S.S., Ten Eyck, L., and Komives, E.A. 2003. Identification of the protein kinase A regulatory RIα‐catalytic subunit interface by amide H/2H exchange and protein docking. Proc. Natl. Acad. Sci. U.S.A. 100:13264‐13269.
   Baerga‐Ortiz, A., Bergqvist, S.P., Mandell, J.G., and Komives, E.A. 2004. Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles. Protein Sci. 13:166‐176.
   Baerga‐Ortiz, A., Hughes, C.A., Mandell, J.G., and Komives, E.A. 2002. Epitope mapping of a monoclonal antibody against human thrombin by H/D‐exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein. Protein Sci. 11:1300‐1308.
   Bai, Y., Milne, J.S., Mayne, L., and Englander, S.W. 1993. Primary structure effects on peptide group hydrogen exchange. Proteins 17:75‐86.
   Dharmasiri, K. and Smith, D.L. 1996. Mass spectrometric determination of isotopic exchange rates of amide hydrogens located on the surfaces of proteins. Anal. Chem. 68:2340‐2344.
   Englander, S. and Mayne, L. 1992. Protein folding studied using hydrogen‐exchange labeling and two‐dimensional NMR. Annu. Rev. Biophys. Biomol. Struct. 21:243‐265.
   Englander, S., Mayne, L., Bai, Y., and Sosnick, T. 1997. Hydrogen exchange: The modern legacy of Linderstrøm‐Lang. Protein Sci. 6:1101‐1109.
   Gemmecker, G., Jahnke, W., and Kessler, H. 1993. Measurement of fast proton exchange rates in isotopically labeled compounds. J. Am. Chem. Soc. 115:11620‐11621.
   Mandell, J.G., Baerga‐Ortiz, A., Akashi, S., Takio, K., and Komives, E.A. 2001. Solvent accessibility of the thrombin‐thrombomodulin interface. J. Mol. Biol. 306:575‐589.
   Mandell, J.G., Falick, A.M., and Komives, E.A. 1998a. Identification of protein‐protein interfaces by decreased amide proton solvent accessibility. Proc. Natl. Acad. Sci. U.S.A. 95:14705‐14710.
   Mandell, J.G., Falick, A.M., and Komives, E.A. 1998b. Measurement of amide hydrogen exchange by MALDI‐TOF mass spectrometry. Anal. Chem. 70:3987‐3995.
   Rosa, J.J. and Richards, F.M. 1979. An experimental procedure for increasing the structural resolution of chemical hydrogen‐exchange measurements on proteins: Application to ribonuclease S peptide. J. Mol. Biol. 133:399‐416.
   Zhang, Z., Post, C.B., and Smith, D.L. 1996. Amide hydrogen exchange determined by mass spectrometry: Application to rabbit muscle aldolase. Biochemistry 35:779‐791.
   Zhang, Z. and Smith, D.L. 1993. Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation. Protein Sci. 2:522‐531.
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