Application of Amide Proton Exchange Mass Spectrometry for the Study of Protein‐Protein Interactions
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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 (D2 O) 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 H2 O 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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- 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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Basic Protocol 1: Identification of the Protein‐Protein Interface Via the On‐Exchange Method
Materials
Support Protocol 1: Analysis of Experimental Data from the On‐Exchange Experiment
Materials
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
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Figure 20.9.1 (at left) Flow chart diagram of the two types of amide proton exchange experiments used to study protein‐protein interfaces. (A ) In the on‐exchange experiment, the protein‐protein complex shows a region in which less deuterium is incorporated when compared with control samples in which each protein is deuterated separately. (B ) In the off‐exchange experiment, each protein is allowed to incorporate deuterium separately, the deuterated proteins are allowed to complex, and then deuterium atoms are off‐exchanged with hydrogen atoms by dilution in H2 O; residues located at the protein‐protein interface are characterized by the retention of deuterons in the protein‐protein complex as compared with a lack of retention in control experiments in which only one of the two proteins is present. View Image -
Figure 20.9.2 (A ) Example of a matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrum of pepsin digest products. The inset shows that the high resolution of the time‐of‐flight mass analyzer allows baseline resolution of the mass envelope for each peptide. Each peptide's mass envelope results from the natural abundance of 13 C within that peptide, such that the peak representing the fraction of peptide molecules containing one 13 C atom appears one mass unit to the right of the monoisotopic peak, the peak representing the fraction containing two 13 C atoms appears two mass units to the right of the monoisotopic peak, and so on. (B ) Sequence coverage map for the protein whose peptide digest mass spectrum is shown in panel A. Each of the lines under the sequence represents a peptide fragment whose mass matches that of a peak observed in the mass spectrum. View Image -
Figure 20.9.3 In the on‐exchange experiment, the interacting proteins, both separately and in the complexed state, are incubated in deuterated buffer for varying lengths of time prior to quenching, pepsin digestion, and mass spectrometric analysis. (A ) Example of a peptide mass envelope resulting from pepsin digestion of (i) nondeuterated CheB protein; (ii) a deuterated protein complex involving CheB; and (iii) CheB alone after being subjected to the same deuteration period as the complex in (ii). The mass envelope broadens somewhat upon deuteration, and there is less deuteration of the peptide in the protein‐protein complex than in the protein alone. (B ) Graph of number of deuterons incorporated into the peptide shown in panel A versus deuteration time. Circles, uncomplexed CheB; squares, complexed CheB. View Image -
Figure 20.9.4 Sample off‐exchange data. A mass envelope for a single peptide fragment at a single off‐exchange time point is shown; however, data from several different time points should be collected to check for consistency in the data. (A ) Nondeuterated peptide fragment from the uncomplexed protein. (B ) Peptide fragment from the uncomplexed protein after on‐exchange for 10 min. (C ) Peptide fragment from the uncomplexed protein after on‐exchange for 10 min followed by off‐exchange for 10 min. Some residual deuteration is seen, due to the fact that some D2 O remains in the H2 O buffer used for off‐exchange. (D ) Peptide fragment from the complexed protein after on‐exchange (performed on each component of the complex separately) for 10 min followed by off‐exchange (performed on the bound complex) for 10 min. In the bound complex, the peptide retains deuterium throughout off‐exchange, whereas in the uncomplexed state, it does not. This indicates that the peptide is probably part of the protein‐protein interface region. View Image -
Figure 20.9.5 Plot showing the relationship between ligand concentration and percentage of receptor molecules bound to the ligand in a typical amide proton exchange experiment. To ensure that 100% of receptor molecules are bound, ligand‐to‐receptor ratios > 1:1 are required when K d ≥ 10 nM. Theoretical curves for K d = 1 nM (circles), K d = 10 nM (squares), and K d = 50 nM (triangles) are shown. Actual experimental data from an interaction for which K d = 120 nM most closely resembled the K d = 50 nM curve. View Image -
Figure 20.9.6 Kinetic plots of amide deuteration after data analysis to correct for back exchange and residual side‐chain deuteration. (A ) On‐exchange data for a solvent‐accessible region of an uncomplexed protein at two different pH values, 6.5 (circles) and 7.9 (squares). (B ) Off‐exchange data for the same solvent‐accessible region in the uncomplexed protein (squares) and in the bound complex (triangles). The data obtained from the uncomplexed protein show that off‐exchange occurs as rapidly as on‐exchange did, and the difference between the two curves indicates that amides are protected from off‐exchange in the complex. Identical data were obtained at both pH 6.5 and pH 7.9. (C ) On‐exchange data for a partially solvent‐accessible region of the uncomplexed protein at two different pH values, 6.5 (circles) and 7.9 (squares). (D ) Off‐exchange data for the same partially accessible region indicate that at pH 6.5, some deuterium is retained in the bound complex (triangles), but only for a short time. No deuterium retention was observed at pH 7.9 (data not shown). View Image
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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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