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Protein Sci. Apr 1993; 2(4): 522–531.
PMCID: PMC2142359

Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.

Abstract

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.

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Selected References

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  • Bentley GA, Delepierre M, Dobson CM, Wedin RE, Mason SA, Poulsen FM. Exchange of individual hydrogens for a protein in a crystal and in solution. J Mol Biol. 1983 Oct 15;170(1):243–247. [PubMed]
  • Brandt P, Woodward C. Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor. Biochemistry. 1987 Jun 2;26(11):3156–3167. [PubMed]
  • Bushnell GW, Louie GV, Brayer GD. High-resolution three-dimensional structure of horse heart cytochrome c. J Mol Biol. 1990 Jul 20;214(2):585–595. [PubMed]
  • Caprioli RM, Fan T. Peptide sequence analysis using exopeptidases with molecular analysis of the truncated polypeptides by mass spectrometry. Anal Biochem. 1986 May 1;154(2):596–603. [PubMed]
  • Delepierre M, Dobson CM, Karplus M, Poulsen FM, States DJ, Wedin RE. Electrostatic effects and hydrogen exchange behaviour in proteins. The pH dependence of exchange rates in lysozyme. J Mol Biol. 1987 Sep 5;197(1):111–130. [PubMed]
  • Englander JJ, Calhoun DB, Englander SW. Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry. Anal Biochem. 1979 Jan 15;92(2):517–524. [PubMed]
  • Englander JJ, Rogero JR, Englander SW. Protein hydrogen exchange studied by the fragment separation method. Anal Biochem. 1985 May 15;147(1):234–244. [PMC free article] [PubMed]
  • Englander SW, Englander JJ, McKinnie RE, Ackers GK, Turner GJ, Westrick JA, Gill SJ. Hydrogen exchange measurement of the free energy of structural and allosteric change in hemoglobin. Science. 1992 Jun 19;256(5064):1684–1687. [PMC free article] [PubMed]
  • Hvidt A, Nielsen SO. Hydrogen exchange in proteins. Adv Protein Chem. 1966;21:287–386. [PubMed]
  • Jeng MF, Englander SW, Elöve GA, Wand AJ, Roder H. Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR. Biochemistry. 1990 Nov 20;29(46):10433–10437. [PubMed]
  • Kossiakoff AA. Protein dynamics investigated by the neutron diffraction-hydrogen exchange technique. Nature. 1982 Apr 22;296(5859):713–721. [PubMed]
  • Loftus D, Gbenle GO, Kim PS, Baldwin RL. Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates. Biochemistry. 1986 Mar 25;25(6):1428–1436. [PubMed]
  • Molday RS, Englander SW, Kallen RG. Primary structure effects on peptide group hydrogen exchange. Biochemistry. 1972 Jan 18;11(2):150–158. [PubMed]
  • Myer YP. Conformation of cytochromes. 3. Effect of urea, temperature, extrinsic ligands, and pH variation on the conformation of horse heart ferricytochrome c. Biochemistry. 1968 Feb;7(2):765–776. [PubMed]
  • Paterson Y, Englander SW, Roder H. An antibody binding site on cytochrome c defined by hydrogen exchange and two-dimensional NMR. Science. 1990 Aug 17;249(4970):755–759. [PMC free article] [PubMed]
  • Roder H, Elöve GA, Englander SW. Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature. 1988 Oct 20;335(6192):700–704. [PMC free article] [PubMed]
  • Roder H, Wagner G, Wüthrich K. Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor. Biochemistry. 1985 Dec 3;24(25):7407–7411. [PubMed]
  • Rosa JJ, Richards FM. An experimental procedure for increasing the structural resolution of chemical hydrogen-exchange measurements on proteins: application to ribonuclease S peptide. J Mol Biol. 1979 Sep 25;133(3):399–416. [PubMed]
  • Smith JB, Sun Y, Smith DL, Green B. Identification of the posttranslational modifications of bovine lens alpha B-crystallins by mass spectrometry. Protein Sci. 1992 May;1(5):601–608. [PMC free article] [PubMed]
  • Takano T, Dickerson RE. Redox conformation changes in refined tuna cytochrome c. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6371–6375. [PMC free article] [PubMed]
  • Thévenon-Emeric G, Kozlowski J, Zhang Z, Smith DL. Determination of amide hydrogen exchange rates in peptides by mass spectrometry. Anal Chem. 1992 Oct 15;64(20):2456–2458. [PubMed]
  • Wagner G, Wüthrich K. Amide protein exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution. Studies with two-dimensional nuclear magnetic resonance. J Mol Biol. 1982 Sep 15;160(2):343–361. [PubMed]
  • Wand AJ, Roder H, Englander SW. Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N-terminal helix. Biochemistry. 1986 Mar 11;25(5):1107–1114. [PubMed]
  • Woodward C, Simon I, Tüchsen E. Hydrogen exchange and the dynamic structure of proteins. Mol Cell Biochem. 1982 Oct 29;48(3):135–160. [PubMed]

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