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Mass Spectrometric Approaches Using Electrospray Ionization Charge States and Hydrogen-Deuterium Exchange for Determining Protein Structures and Their Conformational Changes*

  • Xuguang Yan
    Affiliations
    Departments of Chemistry, Oregon State University, Corvallis, OR 97331
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  • Jeffrey Watson
    Affiliations
    Departments of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
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  • P. Shing Ho
    Affiliations
    Departments of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
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  • Max L. Deinzer
    Correspondence
    To whom correspondence should be addressed. Tel.: 541-737-1773; Fax: 541-737-0497
    Affiliations
    Departments of Chemistry, Oregon State University, Corvallis, OR 97331
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  • Author Footnotes
    * This work was supported by grants from the National Institute on Environmental Health Sciences (ES 00040 and ES 00210) to M. L. D. and from the National Institutes of Health (RIGM 62957A) and National Science Foundation (MCB-0090615) to P. S. H. We acknowledge the support of the nucleic acid and protein core and the mass spectrometry core in the Oregon State University Environmental Health Sciences Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.
      The globular or braided topology with attendant surface crevices and interior cavities creates a unique three-dimensional structure for proteins and, together with their dynamic properties mediated through local fluctuations or large-scale conformational changes, the singular functions of these life-sustaining biomolecules are executed. Conformations and conformational dynamics are key to the protein’s functional integrity and these characteristic properties are highly dependent on environmental conditions. Thus, varying physiological conditions may play an important role in protein conformational changes. For example, pH adjustments in organisms can drive ligand-receptor dissociation and receptor recycling (
      • Clague M.J.
      Molecular aspects of the endocytic pathway..
      ). Protein conformational changes are also manifest in the protein life cycles from expression to final post-activity phase, i.e. birth, function, and death (
      • Fink A.L.
      Compact intermediate states in protein folding..
      ), and improper protein conformations may be responsible for a number of diseases (
      • Parkhomenko T.V.
      • Klicenko O.A.
      • Shavlovski M.M.
      • Kuznetsova I.M.
      • Uversky V.N.
      • Turoverov K.K.
      Biophysical characterization of albumin preparations from blood serum of healthy donors and patients with renal diseases. Part I: Spectrofluorometric analysis..
      ). Detailed information is required on protein higher-order structures and dynamics to fully understand how proteins perform their biological functions at the molecular level.
      Protein conformational changes can be monitored by conventional biophysical and analytical methods, including circular dichroism (CD)
      The abbreviations used are: CD, circular dichroism; AMBER, assisted model building with energy refinement; CID, collision-induced dissociation; CSD, charge-state distribution; ESI, electrospray ionization; H/D, hydrogen/deuterium; MS, mass spectrometry; NMR, nuclear magnetic resonance; rhM-CSF, recombinant human macrophage-colony stimulating factor; TRX, thioredoxin; Oxi-TRX, oxidized thioredoxin; Red-TRX, reduced thioredoxin; GS-Et-TRX, C32-ethylglutathionylated thioredoxin; Cys-Et-TRX, C32-ethylcysteinylated thioredoxin; UV, ultraviolet; LC, liquid chromatography; MS/MS, tandem mass spectrometry.
      1The abbreviations used are: CD, circular dichroism; AMBER, assisted model building with energy refinement; CID, collision-induced dissociation; CSD, charge-state distribution; ESI, electrospray ionization; H/D, hydrogen/deuterium; MS, mass spectrometry; NMR, nuclear magnetic resonance; rhM-CSF, recombinant human macrophage-colony stimulating factor; TRX, thioredoxin; Oxi-TRX, oxidized thioredoxin; Red-TRX, reduced thioredoxin; GS-Et-TRX, C32-ethylglutathionylated thioredoxin; Cys-Et-TRX, C32-ethylcysteinylated thioredoxin; UV, ultraviolet; LC, liquid chromatography; MS/MS, tandem mass spectrometry.
      (
      • Pelton J.T.
      • McLean L.R.
      Spectroscopic methods for analysis of protein secondary structure..
      ), tryptophan fluorescence (
      • Bucci E.
      • Steiner R.F.
      Anisotropy decay of fluorescence as an experimental approach to protein dynamics..
      ), and infrared spectroscopy (
      • Barth A.
      • Zscherp C.
      Substrate binding and enzyme function investigated by infrared spectroscopy..
      ). Far-ultraviolet (UV) CD spectroscopy, for instance, provides information mainly on secondary structural elements of the polypeptide chains; near-UV-CD detects changes in the tertiary structure around aromatic amino acid side chains, and fluorescence techniques may be another more sensitive technique for this purpose. The low-resolving power inherent in these methods, however, limits the investigator’s ability to focus on regions where conformational changes might occur. Detailed structural information at individual residues of a protein can, of course, be obtained from x-ray diffraction (
      • Ringe D.
      • Petsko G.A.
      Mapping protein dynamics by x-ray diffraction..
      ) and nuclear magnetic resonance (NMR) spectroscopy (
      • Ferentz A.E.
      • Wagner G.
      NMR spectroscopy: A multifaceted approach to macromolecular structure..
      • Ohki S.-Y.
      • Eto M.
      • Kariya E.
      • Hayano T.
      • Hayashi Y.
      • Yazawa M.
      • Brautigan D.
      • Kainosho M.
      Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation..
      • Hooke S.D.
      • Radford S.E.
      • Dobson C.M.
      The refolding of human lysozyme: A comparison with the structurally homologous hen lysozyme..
      • Tolman J.R.
      • Flanagan J.M.
      • Kennedy M.A.
      • Prestegard J.H.
      NMR evidence for slow collective motions in cyanometmyoglobin..
      ). However, obtaining a suitable crystal for x-ray diffraction analysis or finding a compatible solvent for proteins, typically less than 30 kDa, to give high enough concentrations for NMR studies is often an obstacle with these methods. Both methods also require relatively large amounts of protein of high purity. Moreover, the conditions required are generally not compatible with the physiological environment of functional proteins, and the static picture of their structures usually is not sufficient for understanding the dynamics associated with performance of their functions.
      The advantages of modern mass spectrometry (MS) for studying protein structure and dynamics are its high mass measuring accuracy and its sensitivity. Electrospray ionization (ESI) MS has become a powerful analytical tool not only to identify proteins and their post-translational modifications but also to analyze their higher-order structures, dynamics, and conformational changes, especially when combined with hydrogen/deuterium (H/D) exchange techniques. H/D exchange coupled with ESI-MS provides a relatively fast and sensitive approach for studying global and regional conformational changes as well as regional dynamics on small amounts of protein.
      The presence of several conformations in equilibrium with different kinetic rates of interchange that depend on environmental conditions represents a typical scenario by which the biological functions may be governed. Thus, the stability, dynamics, and folding pathways of proteins are often studied by examining them under a variety of conditions to mimic protein-native or folded state and/or conformationally altered states. MS is uniquely suited for monitoring co-existing conformations of proteins, regardless of size and physical properties. These are issues that must be addressed when considering x-ray analysis or NMR spectroscopy for similar studies. However, there are potential pitfalls in using MS, and thus it should always be considered as a complementary tool to other well-established biophysical methods for elucidating protein structure and performing conformational analyses. In this article, an attempt is made to summarize some recent developments in methods for determining protein solution phase conformational properties by MS alone or coupled with H/D exchange techniques.

      CONFORMATIONAL CHANGES BY CHARGE-STATE ANALYSIS

      Multiply protonated protein ions are generated in solution and transferred into the gas phase of the mass spectrometer during the ESI process. The observed charge states or charge-state distributions (CSD) of proteins in mass spectra are influenced by various experimental conditions, including pH (
      • Chowdhury S.K.
      • Katta V.
      • Chait B.T.
      Probing conformational changes in proteins by mass spectrometry..
      • Wang G.
      • Cole R.B.
      • Gumerov D.R.
      • Dobo A.
      • Kaltashov I.A.
      Protein-ion charge-state distributions in electrospray ionization mass spectrometry: Distinguishing conformational contributions from masking effects..
      ), temperature (
      • Mirza U.A.
      • Cohen S.L.
      • Chait B.T.
      Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry..
      ,
      • Maier C.S.
      • Schimerlik M.I.
      • Deinzer M.L.
      Thermal denaturation of Escherichia coli thioredoxin studied by H/D exchange and electrospray ionization mass spectrometry: Monitoring a two-state protein unfolding transition..
      ), solvent composition (
      • Wang G.
      • Cole R.B.
      ,
      • Iavarone A.T.
      • Jurchen J.C.
      • Williams E.R.
      Effects of solvent on the maximum charge state and CSD of protein ions produced by electrospray ionization..
      ), and instrument settings (
      • Farmer T.B.
      • Caprioli R.M.
      Electrospray ionization mass spectrometry: Protein structure..
      ). In addition, the intrinsic properties of a protein such as its gas-phase chemistry (
      • Schnier P.D.
      • Gross D.S.
      • Williams E.R.
      On the maximum charge state and proton transfer reactivity of peptide and protein ions formed by electrospray ionization..
      ), sequence (
      • Smith R.D.
      • Loo J.A.
      • Ogorzalek Loo R.R.
      • Busman M.
      • Udseth H.R.
      Principles and practice of electrospray ionization-mass spectrometry for large polypeptides and proteins..
      ) and conformations (
      • Konermann L.
      • Douglas D.J.
      Acid-induced unfolding of cytochrome c at different methanol concentrations: Electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure..
      ,
      • Loo J.A.
      • Edmonds C.G.
      • Udseth H.R.
      • Smith R.D.
      Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra..
      ) are important. The latter contribution to the CSD pattern is the basis for current applications and it may provide an alternative method to CD, for example, for determining overall protein conformational states. However, controversy in using ESI for these application still reigns, particularly with regard to whether there is a correspondence between protein solution and protein gas-phase conformations subsequent to the ESI process and whether different conformations can actually be represented by charge-state envelopes.
      Desorption and transport of multiply charged biomolecules from liquid droplets in the ion source to the gas phase have been studied extensively, but the influence on the three-dimensional structures of proteins during the electrospray process is still unclear. In many cases, ESI mass spectra appear to reflect the expected solution phase conformations rather well (
      • Nesatyy V.J.
      Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry..
      • Wang F.
      • Freitas M.A.
      • Marshall A.G.
      • Sykes B.D.
      Gas-phase memory of solution-phase protein conformation: H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry of the N-terminal domain of cardiac troponin C..
      • Chillier X.F.D.
      • Monnier A.
      • Bill H.
      • Gülaçar F.O.
      • Buchs A.
      • McLuckey S.A.
      • Berkel G.J.V.
      A mass spectrometry and optical spectroscopy investigation of gas-phase ion formation in electrospray..
      • Loo J.A.
      • Loo R.R.O.
      Applying charge discrimination with electrospray ionization-mass spectrometry to protein analyses..
      • Wang G.
      • Cole R.B.
      Mechanistic interpretation of the dependence of CSDs on analyte concentrations in electrospray ionization mass spectrometry..
      • Mao D.
      • Babu K.R.
      • Chen Y.L.
      • Douglas D.J.
      Conformations of gas-phase lysozyme ions produced from two different solution conformations..
      ). For example, the N-terminal domain of cardiac muscle troponin C by Fourier transform ion cyclotron resonance MS (
      • Nesatyy V.J.
      Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry..
      ) exhibited the same charge states and relative intensities when electrosprayed either from water in which the protein was in its native state or from 70% methanol/water/0.25% acetic acid solutions in which it had greater helicity. The identical charge states for the different conformations in these two solutions allowed a direct comparison of the conformations in the gas phase by H/D exchange. From these results, the authors were able to conclude that gas-phase protein ions can retain at least some memory of their solution-phase conformations (
      • Wang F.
      • Freitas M.A.
      • Marshall A.G.
      • Sykes B.D.
      Gas-phase memory of solution-phase protein conformation: H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry of the N-terminal domain of cardiac troponin C..
      ). On the other hand, other studies suggest there is little correspondence (
      • Chillier X.F.D.
      • Monnier A.
      • Bill H.
      • Gülaçar F.O.
      • Buchs A.
      • McLuckey S.A.
      • Berkel G.J.V.
      A mass spectrometry and optical spectroscopy investigation of gas-phase ion formation in electrospray..
      • Loo J.A.
      • Loo R.R.O.
      Applying charge discrimination with electrospray ionization-mass spectrometry to protein analyses..
      • Wang G.
      • Cole R.B.
      Mechanistic interpretation of the dependence of CSDs on analyte concentrations in electrospray ionization mass spectrometry..
      ) between gas- and solution-phase conformations. Thus, gas-phase H/D exchange profiles of lysozyme electrosprayed from water in which the protein should be in its native state, or from water/methanol 20:80 solution in which it should exist as a denatured helical state, showed little difference between the conformations from these two solutions. Apparently, unfolded lysozyme conformations in solution refold to compact ions in the gas phase (
      • Mao D.
      • Babu K.R.
      • Chen Y.L.
      • Douglas D.J.
      Conformations of gas-phase lysozyme ions produced from two different solution conformations..
      ) because of the presence of four intact disulfide bonds that allowed the protein to retain sufficient structure that it refolded quickly in the gas phase prior to deuterium exchange. The pH of the solution from which the protein is electrosprayed is probably important too, as it was reported that at neutral pH the charge-state distributions of α-dendrotoxin reflect the solution structure exactly, but under acidic conditions the solution conformations are only partly retained (
      • Belva H.
      • Lange C.
      Conformational properties of a-dendrotoxin using electrospray mass spectrometry..
      ).
      The relationship between charge states in the gas phase and those in solution has also received a considerable amount of attention (
      • Iavarone A.T.
      • Jurchen J.C.
      • Williams E.R.
      Effects of solvent on the maximum charge state and CSD of protein ions produced by electrospray ionization..
      ,
      • Nesatyy V.J.
      Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry..
      ,
      • Sterner J.L.
      • Johnston M.V.
      • Nicol G.R.
      • Ridge D.P.
      Apparent proton affinities of highly charged peptide ions..
      • Loo J.A.
      • He J.X.
      • Cody W.L.
      Higher order structure in the gas phase reflects solution structure..
      • Loo R.R.O.
      • Smith R.D.
      Investigation of the gas-phase structure of electrosprayed proteins using ion-molecule reactions..
      • Carbeck J.D.
      • Severs J.C.
      • Gao J.
      • Wu Q.
      • Smith R.D.
      • Whitesides G.M.
      Correlation between the charge of proteins in solution and in the gas phase investigated by protein charge ladders, capillary electrophoresis, and electrospray ionization mass spectrometry..
      ). The extent of protonation observed in an ESI spectrum is proposed to be dependent on the steric accessibility of basic amino acids (Lys, Arg, His, and the N terminus) to protonating solvents before entering the gas phase (
      • Covey T.R.
      • Bonner R.F.
      • Shushan B.I.
      • Henion J.J.
      The determination of protein, oligonucleotide and peptide molecular weights by ion-spray mass spectrometry..
      ). Excellent agreement has been observed between the maximum charge states and the numbers of basic residues for many proteins (
      • Smith R.D.
      • Loo J.A.
      • Ogorzalek Loo R.R.
      • Busman M.
      • Udseth H.R.
      Principles and practice of electrospray ionization-mass spectrometry for large polypeptides and proteins..
      ,
      • Covey T.R.
      • Bonner R.F.
      • Shushan B.I.
      • Henion J.J.
      The determination of protein, oligonucleotide and peptide molecular weights by ion-spray mass spectrometry..
      ). Recent studies suggested that the maximum number of charges and the CSDs depend on the relative gas-phase basicities, i.e. the solvents with higher gas-phase basicities have a propensity to remove protons from higher charge state ions and therefore influence the CSDs (
      • Halgand F.
      • Laprevote O.
      Mean charge state and CSD of proteins as structural probes. An electrospray ionisation mass spectrometry study of lysozyme and ribonuclease A..
      ). For the most part, mass spectrometrists have adopted the view that charges on a given protein are not lost during desolvation in the gas phase and that the charge states observed in the gas phase are a reliable indicator of the charges on the protein in solution.
      It is the consensus that ESI mass spectra of proteins in their unfolded states show higher multiply charged ion envelopes (lower m/z value) than the corresponding folded or compact forms (
      • Fenn J.B.
      Ion formation from charged droplets: Roles of geometry, energy, and time..
      ), although there may be exceptions (
      • Mao D.
      • Babu K.R.
      • Chen Y.L.
      • Douglas D.J.
      Conformations of gas-phase lysozyme ions produced from two different solution conformations..
      ). In solution, proteins fold and unfold continuously, even under conditions that approach those of physiological environments, i.e. temperature, pH, and ionic strength, resulting in the continuous presence of transitional intermediates (
      • Kuloglu E.S.
      • McCaslin D.R.
      • Markley J.L.
      • Volkman B.F.
      Structural rearrangement of human lymphotactin, a C chemokine, under physiological solution conditions..
      ). However, a favored conformation governed by environmental conditions may represent the bulk of the protein population. The analysis of protein CSD patterns in ESI spectra is generally considered to produce only qualitative, low-resolution information. The numbers of conformational states may not be resolved by the numbers of charge-state envelopes in this straightforward method because the same degree of charging may occur with different conformational states (
      • Kaltashov I.A.
      • Eyles S.J.
      Studies of biomolecular conformations and conformational dynamics by mass spectrometry..
      ). The mathematical deconvolution of the charge-state envelopes may help to enhance the structural details. Thus, the evolution of partially unfolded intermediates along with the folded and unfolded forms for holo-myoglobin and apo-myoglobin as a function of pH could be studied by ESI charge-state envelopes (
      • Dobo A.
      • Kaltashov I.A.
      Detection of multiple protein conformational ensembles in solution via deconvolution of charge-state distributions in ESI MS..
      ).
      In practice, the monitoring of transitions between the native and denatured states of a protein in solution by charge-state envelope analysis can be very useful if the system is relatively uncomplicated, i.e. a two-state folding/unfolding model (
      • Privalov P.L.
      ) with no intermediate states. Most single-domain proteins are believed to approach closely this ideal (
      • Creighton T.E.
      Protein folding..
      ), and CSDs can be used to determine the ratio of folded and unfolded states as well as the kinetics of unfolding and protein stability. Thioredoxin (TRX), for example, has 13 basic sites distributed throughout its 108-aa residue sequence that are possible sites for protonation. The maximum charges observed in the ESI spectrum for unfolded TRX is 15 (Fig. 1 ). Clearly, there are at least two other sites with sufficiently high proton affinity to be observed under ESI conditions. The analyses of TRXs (oxidized [Oxi-TRX], reduced [Red-TRX], C32-ethylglutathionylated [GS-Et-TRX], and C32-ethylcysteinylated [Cys-Et-TRX]) by ESI charge-state envelopes produced melting curves that provided substantial information on the structures of these proteins (Fig. 2 A) (
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      ). The results indicate that Oxi-TRX is most stable (Tm = 67 °F) while the reduced form and its derivatives show similar stabilities with melting temperature Tm ∼ 54 °C. There is reasonable agreement in the stability order of TRXs, but the melting temperatures are 3–10 °C lower by near-UV-CD than when measured by ESI charge-state envelopes (Fig. 2B). The near-UV-CD signal represents the total of four aromatic amino acids in TRX, i.e. two tryptophans in the active site region and two in α helices remote from the active site. The gradual decrease in molar ellipticity is likely due to minor changes in the asymmetric environment near the tryptophans and tyrosines as a result of increased rotational and vibrational motions as the temperature is increased. The mass spectrometrically derived melting curves remain constant up to the transition point because such low-energy motions do not affect the numbers of charges on the protein. Increased protonation occurs when basic sites become available through unfolding near the melting temperature.
      Figure thumbnail gr1
      Fig. 1ESI mass spectra of Oxi-TRX in 1% acetic acid recorded at different temperatures. The charge states +7 to +9 were attributed to the folded form and hence marked F, and charge states +10 to +15 were attributed to the unfolded form and thus marked U. (Adapted with permission from Ref.
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      .)
      Figure thumbnail gr2
      Fig. 2Heat denaturation curves of four TRX forms in 1% acetic acid. Analysis performed by (A) charge-state distributions in ESI-MS spectra and (B) ellipticity in near-UV-CD spectra. The arrows indicate the melting temperature for each derivative. (Adapted with permission from Ref.
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      .)
      There is significance to the fact that Red-TRX and Cys-Et-TRX show similar melting curves, whereas GS-Et-TRX has an apparent folded/unfolded composition more like that of Oxi-TRX in the temperature region T < Tm, but nearly identical to that of Red-TRX in the region T > Tm. Modeling of GS-Et-TRX by assisted model building with energy refinement (AMBER) force field suggested that two salt bridges are formed between the carboxylates of the γ-glutamyl and glycylyl residues of the glutathionyl moiety and the basic residue side chains of Arg73 and Lys90, thereby imposing structural compactness and restriction to protonation in the folded form (
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Ho P.S.
      • Deinzer M.L.
      Intramolecular interactions in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      ). Such salt bridges are not present in Cys-Et-TRX, and the modification has no greater influence on the melting curve than that observed in the reduced from, i.e. Red-TRX.
      Thus, it can be concluded that the charge-state envelopes reflect the overall conformational properties of these proteins whereas near-UV-CD is more descriptive of localized environmental conditions. The dramatic changes among the different temperature-dependent ellipticity profiles of TRXs appear to be most affected by changes in the active site region. The near superimposeability of the GS-Et- and Cys-Et-TRX profiles reflect common environmental conditions with changing temperature regardless of the nature of the alkylation, but the unusual profile for Red-TRX points to a more fundamental phenomenon. Generally, when melting profiles are not superimposable, transient intermediates are suspected. Indeed, H/D exchange in conformations above the Tm in the unfolding profiles showed minor peaks between the protonated and deuterated peaks (
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      ), indicating that none of the TRXs follow a true cooperative two-state unfolding model.
      Organic solvent- or pH variation-induced protein unfolding can be observed by CSD analysis as demonstrated with ferricytochrome c (
      • Konermann L.
      • Douglas D.J.
      Acid-induced unfolding of cytochrome c at different methanol concentrations: Electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure..
      ). In 3% methanol near neutral pH, cytochrome c displayed a native folded conformation with an envelope centered on the +8 charge state (Fig. 3 A). At pH 4.2, the protein apparently retained a native-like conformation as indicated by the ESI spectrum but the envelope is now centered on the +9 charge state (Fig. 3B), indicating perhaps some loosening or a “secondary” solvent effect, but no real unfolding. A distinct second envelope is centered about the +17 charge state for the unfolded state at lower pH (Fig. 3, C and D). In 50% methanol and pH 3.8, the appearance of the folded charge-state envelope centered at +10, and an unfolded state at +14, indicated there was a different conformation present (Fig. 3E). Far-UV-CD showed retention of secondary structure in 50% methanol, whereas both tertiary and secondary structural breakdown occurred under acidic conditions in 3% methanol. This is a clear illustration that ESI charge-state envelopes can detect the presence of several protein conformers under a given set of experimental conditions.
      Figure thumbnail gr3
      Fig. 3ESI mass spectra of cytochrome c in water containing 3% methanol and 0.5 mm ammonium acetate.A, pH 6.4; B, pH 4.2; C, pH 2.6; D, pH 2.3; and E, in 50% methanol and 0.5 mm ammonium acetate at pH 3.8. (Reprinted with permission from Ref.
      • Konermann L.
      • Douglas D.J.
      Acid-induced unfolding of cytochrome c at different methanol concentrations: Electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure..
      .)
      ESI-MS charge-state envelopes were also used to show the collapse of the tertiary structure of Zn2+ complex colicin E9 endonuclease upon acid-induced release of the metal ion. Moreover, these studies revealed that the noncovalent protein-protein complexes, i.e. colicin E9 endonuclease and its cognate immunity protein, Im9, dissociate in the gas phase before the metal ion complex does, thereby confirming that electrostatics and not hydrophobic interactions are more important in the gas phase (
      • Bremer E.T.J.V.D.
      • Jiskoot W.
      • James R.
      • Moore G.R.
      • Kleanthous C.
      • Heck A.J.R.
      • Maier C.S.
      Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry..
      ). Another example of the power of charge-state analysis to elucidate structural properties of a protein where other methods fail is illustrated by studies on human recombinant macrophage-colony stimulating factor β (rhM-CSFβ) (
      • Zhang Y.H.
      • Yan X.
      • Maier C.S.
      • Schimerlik M.I.
      • Deinzer M.L.
      Structural comparison of recombinant human macrophage colony stimulating factor b and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry..
      ). This compact homodimer with nine disulfide bonds exhibits a charge-state envelope centered on +21 with a maximum of +29 charges (Fig. 4 A). Selective reduction of two disulfide bonds (C157, 159–C′157, 159) near the C-terminal tail showed little change in the secondary structure as suggested by far-UV-CD and fluorescence spectra, and even the three-dimensional integrity of the protein was unaltered as suggested by the unchanged biological activity. However, the ESI spectrum clearly revealed there were major changes as the charge states increased to a maximum of +41 (Fig. 4B), suggesting that six additional basic sites became accessible in each subunit by removal of two disulfide bonds. However, only Lys154 would appear to become available for protonation upon reduction of the disulfides, C157, 159–C′157, 159, as all other basic sites are in the N-terminal direction relative to the next stabilizing disulfide bond, C102–C146. Apparently, an overall loosening of the structure occurred as will be discussed later, thereby causing greater exposure of additional basic residues to the solvent.
      Figure thumbnail gr4
      Fig. 4Positive ion ESI mass spectra of rhM-CSFβ sprayed from acetonitrile/H2O containing 0.1% trifluoroacetic acid solution through a column.A, Native state; and B, selectively reduced and blocked sulfhydryls C157–C159.
      Clearly ESI charge-state envelopes yield considerable information when proteins are denatured thermally or under acidic or varying solvent conditions and when chemical modifications are made as in the reduction of disulfide bonds. In fact, the method has advantages over CD because specific chromophores on the protein are not required and multiple interactions and conformational changes can be observed simultaneously. The tertiary structure and changes to the structure are the important features of proteins in the performance of their natural functions, and the simple but sensitive ESI charge-state envelopes can fortuitously reveal some of the details. Although ESI experiments can reveal structural changes in proteins affected by temperature, pH, and disulfide cleavages, the effects of denaturing agents, urea, and guanidine hydrochloride on protein unfolding or conformations cannot be determined directly by this method because of the intolerance of ESI for salts. When examining conformations or conformational dynamics under the influence of chemical denaturants, CD may be the better method, but such conformational changes can be determined by liquid chromatography (LC) MS in combination with H/D exchange techniques performed both off-line and on-line.

      CONFORMATIONAL CHANGES BY SITE-SPECIFIC STRUCTURAL ANALYSIS

      If ESI-MS has the capability to probe protein conformational dynamics through analysis of CSDs that is at least as good as any other method, then the power of MS to probe conformational changes of proteins is advanced still further when H/D exchange and protein fragmentation techniques are applied. While charge-state analysis provides information on the overall compactness of the protein, regional or site-specific amide hydrogen exchange can be used to probe localized conformations and conformational dynamics. Generally, the level of exchanged hydrogens reflects the relative openness, solvent accessibility, or hydrogen bonding strength in protein structures (
      • Wagner D.S.
      • Anderegg R.J.
      Conformation of cytochrome c studied by deuterium exchange-electrospray ionization mass spectrometry..
      • Babu K.R.
      • Moradian A.
      • Douglas D.J.
      Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry..
      • Eyles S.J.
      • Dresch T.
      • Gierasch L.M.
      • Kaltashov I.A.
      Unfolding dynamics of a beta-sheet protein studied by mass spectrometry..
      • Miranker A.
      • Robinson C.V.
      • Radford S.E.
      • Dobson C.M.
      Investigation of protein folding by mass spectrometry..
      ).
      The H/D exchange method was introduced by Linderstrom-Lang and coworkers four decades ago (
      • Hvidt A.
      • Linderstrom-Lang K.
      Exchange of hydrogen atoms in insulin with deuterium atoms in aqueous solutions..
      ). Since then, many fundamental studies have been undertaken directed at understanding protein dynamics using various analytical techniques to measure hydrogen isotope exchange. Among them, NMR has played a central role and continues to do so as newer high-field instruments and chromatographic techniques are applied. In the late seventies, hydrogen isotope exchange experiments involved tritium labeling and radiation counting to probe protein structure and dynamics (
      • Rosa J.J.
      • Richards F.M.
      An experimental procedure for increasing the structural resolution of chemical hydrogen-exchange measurements on proteins: Application to ribonuclease S peptide..
      ). The techniques were developed further by Englander and coworkers, with low temperature (0 °C) and adjusted pH (2.4–2.8) to minimize the loss of isotopic label during the high-performance LC step (
      • Englander J.J.
      • Rogero J.R.
      • Englander S.W.
      Protein hydrogen exchange studied by the fragment separation method..
      ). Deuterium was substituted for tritium when NMR, and later MS, emerged as tools in the analyses of H/D levels. MS provided a particularly important dimension as it allowed measuring accurate molecular masses of fragmented peptides and proteins, thereby providing information on the numbers of hydrogens that had been exchanged.
      When protonated proteins are solubilized in a large excess of deuterated solvent, the hydrogens on proteins including those at amide sites, N and C termini, and other functionalities, e.g. in side chains containing oxygen, nitrogen, or sulfur, undergo exchange. The rate of exchange of amide hydrogens varies over a range 108 depending mostly on their solvent accessibility and hydrogen bonding status, while hydrogens on functional groups exchange too fast to be measured conveniently by most techniques. These back exchange quickly when exposed to excess protic solvents as, for example, during high-performance LC separation, but amide hydrogen isotopes remain (
      • Englander J.J.
      • Rogero J.R.
      • Englander S.W.
      Protein hydrogen exchange studied by the fragment separation method..
      ). The information on protein backbones is then provided by the presence of the hydrogen isotope on the amide nitrogens.
      The application of MS to measure H/D content in peptides and proteins was first introduced by Katta and Chait in 1991 (
      • Katta V.
      • Chait B.T.
      Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry..
      ). The mechanism of amide hydrogen exchange, which can be either acid- or base-catalyzed (
      • Eigen M.
      Proton transfer, acid-base catalysis, and enzymic hydrolysis..
      ), is well understood from experiments performed with extended, unstructured peptides in solution. The exchange reaction is slowest at pH 2–3, which is about 103–104 times slower as compared with the rate around pH 7 (
      • Bai Y.
      • Milne J.S.
      • Mayne L.
      • Englander S.W.
      Primary structure effects on peptide group hydrogen exchange..
      ,
      • Smith D.L.
      • Deng Y.
      • Zhang Z.
      Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry..
      ). The exchange reaction rate is also temperature-dependent, decreasing by about a factor of 10 as the temperature is reduced from 25 to 0 °C. Generally, under pH 2–3 and 0 °C, which is commonly referred to as “quench conditions,” the half-life for amide hydrogen isotopic exchange in an unstructured polypeptide is 30–90 min. This sensitivity to pH and temperature is the basis for conducting exchange under a given set of conditions and arresting it under another, thereby allowing the sample to be prepared for analysis by ESI-MS or other mass spectrometric approaches. It should be noted that the mechanism of amide hydrogen isotope exchange in the gas phase is thought to be very different from that in solution phase, making it difficult, if not unrealistic, to compare the processes in the two phases (
      • Freitas M.A.
      • Hendrickson C.L.
      • Emmett M.R.
      • Marshall A.G.
      Gas-phase bovine ubiquitin cation conformations resolved by gas-phase H/D exchange rate and extent..
      • Campbell S.
      • Rodgers M.T.
      • Marzluff E.M.
      • Beauchamp J.L.
      Deuterium exchange reactions as a probe of biomolecule structure. Fundamental studies of gas phase H/D exchange reactions of protonated glycine oligomers with D2O, CD3OD, CD3CO2D, and ND3..
      • Wyttenbach T.
      • Bowers M.T.
      Pressure limited sustained off-resonance irradiation for collision-activated dissociation in Fourier transform mass spectrometry..
      • Freitas M.A.
      • Hendrickson C.L.
      • Emmett M.R.
      • Marshall A.G.
      Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry..
      ).
      Over the past three decades, H/D analysis by NMR, which also measures amide hydrogen isotope levels, has been the single most powerful approach for determining protein conformation and dynamics in solution (
      • Ferentz A.E.
      • Wagner G.
      NMR spectroscopy: A multifaceted approach to macromolecular structure..
      ,
      • Englander J.J.
      • Englander S.W.
      • Louie G.
      • Roder H.
      • Tran T.
      • Wand A.J.
      ). It has, in fact, become the standard for comparison when other techniques are applied. Recently, however, H/D analysis by MS has proved to be complementary to NMR and x-ray crystallographic methods for studying protein conformations and conformational changes. Dobson and coworkers were the first to show the unique advantages of ESI-MS analysis of hydrogen isotope ratios following pulse labeling when they performed a nonequilibrium time course study on the refolding pathway of hen egg-white lysozyme (
      • Miranker A.
      • Robinson C.V.
      • Radford S.E.
      • Aplin R.T.
      • Dobson C.M.
      Detection of transient protein folding populations by mass spectrometry..
      ). Their studies showed fully protected, unprotected, or partially protected transient folding intermediates that, together with NMR rate data, were interpreted as involving cooperative folding of α and β domains in which the α domain folds independently in an initial fast phase, and a sequential folding of the α and then the β domain follows in slower phases. NMR data alone could not have distinguished between this mechanism and one involving independent folding pathways for the two domains in the fast kinetic phase because the technique measures labeling isotopic levels averaged over all populations of protein in the sample. Further studies that coupled hydrogen exchange and MS proved that the α domain is destabilized both at high temperature (50 °C) (
      • Matagne A.
      • Jamin M.
      • Chung E.W.
      • Robinson C.V.
      • Radford S.E.
      • Dobson C.M.
      Thermal unfolding of an intermediate is associated with non-arrhenius kinetics in the folding of hen lysozyme..
      ) and when one of the disulfide bonds (C6–C127) is reductively alkylated (
      • Eyles S.J.
      • Radford S.E.
      • Robinson C.V.
      • Dobson C.M.
      Kinetic consequences of the removal of a disulfide bridge on the folding of hen lysozyme..
      ).
      Smith and coworkers (
      • Zhang Z.
      • Smith D.L.
      Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation..
      ) established a general procedure (Fig. 5) for protein H/D exchange and digestion experiments in applications that utilize ESI-MS for analysis. This technique made it possible to probe protein conformational changes with a spatial resolution of better than 5–10 residues when not limited by proteolytic cleavages. Overall, deuterium levels in peptic peptides can be obtained from the increase in molecular mass after exchange-in. The centroids of the isotopic clusters are used to determine the backbone amide site H/D content, and the deuterium levels at individual amide sites can in principle be obtained in the same way when collision-induced dissociation (CID) tandem MS (MS/MS) is used for sequencing.
      Figure thumbnail gr5
      Fig. 5General procedure used for H/D exchange MS experiments established by Smith and coworkers (
      • Zhang Z.
      • Smith D.L.
      Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation..
      ). Deuterium exchanges into intact protein during the labeling period. Labeling is quenched by decreasing the pH to 2–3 and temperature to 0 °C. Deuterium levels at peptide amide linkages in either the intact protein (global exchange profile) or in peptic fragments (local exchange profile) are determined by LC-ESI-MS.
      CID MS/MS-fragmented peptides, dominated by cleavage at the peptide bonds resulting in bn and yn ions, yield the sequence information and potentially the H/D content at individual amide sites. A reluctance to relying too heavily on CID MS/MS to measure the isotopic content at individual amide linkages after hydrogen isotope exchange stems from studies that showed scrambling of the label during the collision process. Thus, significant hydrogen scrambling was observed during H/D analysis on cytochrome c after activation by sustained off-resonance irradiation in the ion cyclotron resonance cell of a mass spectrometer (
      • McLafferty F.W.
      • Guan Z.
      • Haupts U.
      • Wood T.D.
      • Kelleher N.L.
      Gaseous conformational structures of cytochrome c..
      ). However, other studies (
      • Miranker A.
      • Robinson C.V.
      • Radford S.E.
      • Dobson C.M.
      Investigation of protein folding by mass spectrometry..
      ,
      • McLafferty F.W.
      • Guan Z.
      • Haupts U.
      • Wood T.D.
      • Kelleher N.L.
      Gaseous conformational structures of cytochrome c..
      ,
      • Demmers J.A.A.
      • Haverkamp J.
      • Heck A.J.
      • Koeppe R.E.
      • Killian J.A.
      Electrospray ionization mass spectrometry as a tool to analyze H/D exchange kinetics of transmembrane peptides in lipid bilayers..
      ) suggested that scrambling was minimal for short helical peptides under typical CID conditions in triple quadrupole instruments. Similarly, there appeared to be no evidence for hydrogen scrambling during fragmentation of transmembrane peptides during CID in a hybrid (Q-TOF) mass spectrometer, but subsequent model studies of transmembrane peptides incorporated in a lipid bilayer indicated that the extent of scrambling was dependent on the nature of the charge carrier and amino acid sequence (
      • Demmers J.A.A.
      • Haverkamp J.
      • Heck A.J.
      • Koeppe R.E.
      • Killian J.A.
      Electrospray ionization mass spectrometry as a tool to analyze H/D exchange kinetics of transmembrane peptides in lipid bilayers..
      ,
      • Demmers J.A.A.
      • Duijn E.v.
      • Haverkamp J.
      • Greathouse D.V.
      • Roger E. Koeppe I.
      • Heck A.J.R.
      • Killian J.A.
      Interfacial positioning and stability of transmembrane peptides in lipid bilayers studied by combining H/D exchange and mass spectrometry..
      ). Alkali-cationized peptide ions showed less scrambling than protonated ions (
      • Demmers J.A.A.
      • Rijkers D.T.S.
      • Haverkamp J.
      • Killian J.A.
      • Heck A.J.R.
      Factors affecting gas-phase deuterium scrambling in peptide ions and their implications for protein structure determination..
      ). Complete scrambling of label was reported when peptides from a study to determine the binding interface between urokinase plasminogen activator and its cellular activator were collisionally activated in an attempt to map the specific binding residues (
      • Jorgensen T.J.D.
      • Ploug M.
      • Roepstorff P.
      ). Thus, although there are examples where mapping of site-specific events by amide H/D analysis have apparently been quite successful, all experimental results involving CID MS/MS in such studies must be viewed with skepticism until this issue is resolved.
      Ion formation studies suggest that the production of yn ions during CID depend on the transfer of a proton from the leaving group, while the bn ions form a protonated oxazolodone structures without proton transfer (Fig. 6), i.e. the amide site to which the proton was originally transferred becomes the leaving group and is of no further consequence in the analysis (
      • Schlosser A.
      • Lehmann W.
      Five-membered ring formation in unimolecular reactions of peptides: A key structural element controlling low-energy collision-induced dissociation of peptides..
      ,
      • Harrison A.G.
      • Siu K.W.M.
      • Aribi H.E.
      Amide bond cleavage in deprotonated tripeptides: A newly discovered pathway to “b2 ions..
      ). Yet, bn and yn ion formation via the “mobile proton model” may still constitute proton migration from a protonated amino acid side chain to carboxyl oxygens and amide nitrogen (
      • Harrison A.G.
      • Yalcin T.
      Proton mobility in protonated amino acids and peptides..
      ). In fact, proton transfer from the side chains of basic amino acids to the less-basic amide nitrogen of the peptide bond (
      • Dongre A.R.
      • Jones J.L.
      • Somogyi A.
      • Wysocki V.H.
      Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: Evidence for the mobile proton model..
      ) may be a necessary step for creating the labile bonds that result in the production of bn ions. The scrambling problem has been attributed partly to the conditions used for CID processes, i.e. the applied collision energy and the time scale for ion activation and fragmentation (
      • Eyles S.J.
      • Speir J.P.
      • Kruppa G.H.
      • Gierasch L.M.
      • Kaltashov I.A.
      Protein conformational stability probed by Fourier transform ion cyclotron resonance mass spectrometry..
      ). Space charge limits may also cause problems by shifting masses during analysis (
      • Voyksner R.D.
      • Lee H.
      Investigating the use of an octupole ion guide for ion storage and high-pass mass filtering to improve the quantitative performance of electrospray ion trap mass spectrometry..
      ). Rapid activation to high energy states from collision processes may reduce the extent of hydrogen scrambling (
      • Kaltashov I.A.
      • Eyles S.J.
      Crossing the phase boundary to study protein dynamics and function: Combination of amide hydrogen exchange in solution and ion fragmentation in the gas phase..
      ). Deuterium levels at individual peptide amide linkages after exchange-in onto proteins as determined through CID MS/MS-generated bn ions have been found to correlate with data derived from NMR exchange rates (
      • Kim M.Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Site-specific amide H/D exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry..
      ,
      • Deng Y.
      • Pan H.
      • Smith D.L.
      Selective isotope labeling demonstrates that hydrogen exchange at individual peptide amide linkages can be determined by collision-induced dissociation mass spectrometry..
      ). The key to successfully measuring H/D content through CID may depend on the mechanism and experimental conditions for formation of the bn ions, but yn ions have appeared to be particularly unreliable for H/D analysis in almost all studies (
      • Deng Y.
      • Pan H.
      • Smith D.L.
      Selective isotope labeling demonstrates that hydrogen exchange at individual peptide amide linkages can be determined by collision-induced dissociation mass spectrometry..
      ).
      Figure thumbnail gr6
      Fig. 6Possible mechanism for formation of b ions.A, protonation of the amide site may proceed via the “mobile hydrogen” prior to formation of the oxazoladone that leads to the b ions. B, the b ions may result from the oxazoladone in which the protonated amide nitrogen becomes the leaving group (
      • Schlosser A.
      • Lehmann W.
      Five-membered ring formation in unimolecular reactions of peptides: A key structural element controlling low-energy collision-induced dissociation of peptides..
      ).
      The information gained from site-specific amide H/D exchange for an entire protein, i.e. TRX, demonstrates the potential of this technique (
      • Kim M.Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Site-specific amide H/D exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry..
      ). Deuterium equilibrium exchange-in experiments with oxidized, reduced, and modified TRXs indicated resistance to exchange in the order observed by F/(F + U) values in CSD (
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      ). However, the influence on protein stability by the two alkyl groups, compared with Red-TRX conformation, are different, as suggested by site-specific amide hydrogen isotopic exchange (
      • Kim M.Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Site-specific amide H/D exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry..
      ). The ethylcysteinyl group in Cys-Et-TRX results in faster exchange at Thr66 and Ala67 than in both Red-TRX and GS-Et-TRX. The ethylcysteinyl group, although small, appears to perturb the conformation of Red-TRX globally as these two sites are distant both in sequence and spatially from the modified Cys32 site. The ethylglutathionyl group in GS-Et-TRX, on the other hand, caused an increase in exchange at Lys90 and Val91, but a decrease at Ile75, Gly92, and Ala93. The difference in behavior between the two alkylated structures is attributed to the salt bridges in GS-Et-TRX mentioned above, which results in a net increase in electrostatic stability calculated to be ∼35 kcal relative to Red-TRX. Induced hydrogen bonding interactions also were predicted between carbonyl oxygens of the conformationally restricted ethylglutathionyl group amide hydrogens in the protein backbone, and H/D data provided strong evidence in support (
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Ho P.S.
      • Deinzer M.L.
      Intramolecular interactions in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      ).
      H/D measurements by LC-ESI-MS/MS analysis must take into account the inevitable back-exchange during LC separation if absolute values are needed. Corrections are made by assuming that deuterium loss throughout is constant with its measured level in comparison to the overall exchange in the control sample according to (
      • Zhang Z.
      • Smith D.L.
      Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation..
      ):
      D=[(mtm0%)/(m100%m0%)]×N
      (Eq. 1)


      where D is the deuterium content of a peptide with N peptide amide hydrogens and mt, m0%, and m100% are peptide molecular masses centroided for H/D exchange-in at t min, 0 min, and fully exchanged, respectively. Serious back-exchange may result in a failure to detect subtle isotopic differences and the ability to conclude anything about a protein’s conformational changes (
      • Yan X.
      • Broderick D.
      • Leid M.E.
      • Schimerlik M.I.
      • Deinzer M.L.
      Dynamics and ligand-induced solvent accessibility changes in human retinoid X receptor homodimer determined by hydrogen deuterium exchange and mass spectrometry.
      ). In addition, complete sequence information from CID MS/MS and at the ends of peptic fragments is often difficult to obtain reliably. Overlapping fragments can be used to increase spatial resolution, but selective back-exchange sometimes causes uncertainty. These problems may be solved if intact protein solutions are sprayed directly into the mass spectrometer. Akashi and co-workers used high-resolution and high-accuracy ESI-Fourier transform ion cyclotron resonance MS to analyze a small labeled protein (8.5 kDa) by capillary-skimmer CID. Less back-exchange (3–4%) was observed in comparison to the 10–30% back-exchange that generally occurs by proteolysis and LC separation of the resulting peptides. The spatial resolution achieved in these studies was consistent with that obtained by NMR and x-ray studies (
      • Alkashi S.
      • Naito Y.
      • Takio K.
      Observation of hydrogen-deuterium exchange of ubiquitin by direct analysis of electrospray capillary-skimmer dissociation with Fourier transform ion cyclotron resonance mass spectrometry..
      ).

      CONFORMATIONAL CHANGES BY EXCHANGE RATE ANALYSIS

      Although amide hydrogen exchange has long been studied and has been widely recognized as a powerful tool to probe protein conformational changes (
      • Hvidt A.
      • Nielsen S.O.
      Hydrogen exchange in proteins..
      • Englander J.J.
      • Calhoun D.B.
      • Englander S.W.
      Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry..
      • Woodward C.
      • Simon I.
      • Tuchsen E.
      Hydrogen exchange and the dynamic structure of proteins..
      • Englander S.W.
      • Mayne L.
      Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR..
      • Clarke J.
      • Itzhaki L.S.
      Hydrogen exchange and protein folding..
      • Raschke T.
      • Marqusee S.
      Hydrogen exchange studies of protein structure..
      ) and the chemical process of exchange in unstructured peptides is quite clear, the mechanisms of exchange in folded proteins are still poorly understood. Proteins are continuously unfolding and refolding even under physiological conditions. Amide hydrogens may exchange directly from the folded state, after global unfolding, or from both according to a commonly accepted model (
      • Hvidt A.
      • Nielsen S.O.
      Hydrogen exchange in proteins..
      ,
      • Bahar I.
      • Wallqvist A.
      • Covell D.G.
      • Jernigan R.L.
      Correlation between native-state hydrogen and cooperative residue fluctuations from a simple model..
      ):
      MathJax Formula fx1
      (Eq. 2)


      where k1 and k−1 are the rate constants for unfolding and refolding, and kint is the exchange rate at this peptide in the random coil conformation. The observed exchange rate (kex) is the sum of contributions of exchange from the folded state (kf) and the globally unfolding state (ku):
      kex=kr+ku
      (Eq. 3)


      The exchange rate constant from the folded state is given by:
      kf=βkint
      (Eq. 4)


      where β is the probability that amide hydrogens contact solvent and catalyst. The exchange rate constant after unfolding is given by:
      ku=k1kint/(k1+kint)
      (Eq. 5)


      When k−1kint, the folding-refolding process occurs many times before H/D exchange is complete. This limiting case is referred to as EX2 kinetics and kex = kint (k1/k−1). When kintk−1, exchange occurs by the EX1 mechanism and kex = k1. When H/D exchange occurs by the EX2 mechanism, which generally describes the exchange in the native state where regional unfolding may occur or under relatively mild denaturing conditions, the mass spectrum shows a single mass peak that gradually shifts to higher mass with increasing exchange-in time (
      • Bai Y.
      • Milne J.S.
      • Mayne L.
      • Englander S.W.
      Protein stability parameters measured by hydrogen exchange..
      ). In contrast, two distinct mass peaks, one that is protonated and one that is largely deuterated, develop after short exchange-in time if exchange occurs via the EX1 mechanism. This mechanism is operative when global unfolding takes place, for example, at high temperatures, under acidic pH conditions, or in the presence of high denaturant concentrations (
      • Maier C.S.
      • Schimerlik M.I.
      • Deinzer M.L.
      Thermal denaturation of Escherichia coli thioredoxin studied by H/D exchange and electrospray ionization mass spectrometry: Monitoring a two-state protein unfolding transition..
      ,
      • Kim M.-Y.
      • Maier C.S.
      • Reed D.J.
      • Deinzer M.L.
      Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
      ,
      • Smith D.L.
      • Deng Y.
      • Zhang Z.
      Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry..
      ). In contrast to EX2 kinetics, the rates of unfolding can be determined within the EX1 kinetic limit.
      The pioneering studies of Dobson, Robinson, and coworkers demonstrated that EX1 and EX2 kinetics in H/D exchange studies could be monitored using MS (
      • Miranker A.
      • Robinson C.V.
      • Radford S.E.
      • Aplin R.T.
      • Dobson C.M.
      Detection of transient protein folding populations by mass spectrometry..
      ). Simply measuring the width of the protein’s mass peak provided information on whether exchange followed EX2 or kinetics intermediate between the two limits. Proteins may undergo isotopic exchange simultaneously by EX1 kinetics in certain regions and EX2 kinetics others. For example, oxidized and reduced TRX in 1% AcOD/D2O at room temperature underwent H/D exchange by EX2 kinetics at the N-terminal end (aa 28–39) and by EX1 type kinetics at the C-terminal end (aa 59–80 and aa 81–101) (Fig. 7) (

      Kim, M.-Y.(2000)Mass Spectrometric Studies on Peptides and Proteins: Conformations of Escherichia coli Thioredoxin and its Alkylated Adducts Studied by H/D Exchange and HPLC-Electrospray Ionization Mass Spectrometry. Ph. D. thesis, Oregon State University

      ). In an elegant series of experiments, pulse-labeling hydrogen isotope exchange followed by ESI-MS analysis of a mixture in the 15N-labeled wild-type human lysozyme and the amyloidogenic D67H mutant showed EX2 exchange for the former and EX1 kinetics in the latter (
      • Canet D.
      • Last A.M.
      • Tito P.
      • Sunde M.
      • Spencer A.
      • Archer D.B.
      • Redfield C.
      • Robinson C.V.
      • Dobson C.M.
      Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme..
      ). The results provided evidence for cooperative unfolding of the β domain and adjacent C helix, allowing the authors to conclude that the extensive intermolecular interactions possible can lead to aggregation and fibrallar structures in patients with the mutant gene.
      Figure thumbnail gr7
      Fig. 7ESI mass spectra of the doubly charged molecular ions of fragment 28–39, the triply charged molecular ions of fragment 59–80, and fragment 81–101 derived from nondeuterated (0% ref), partially deuterated, and completely deuterated (100% ref) Oxi-TRX. (Reprinted with permission from Ref.

      Kim, M.-Y.(2000)Mass Spectrometric Studies on Peptides and Proteins: Conformations of Escherichia coli Thioredoxin and its Alkylated Adducts Studied by H/D Exchange and HPLC-Electrospray Ionization Mass Spectrometry. Ph. D. thesis, Oregon State University

      .)
      Two types of structural models have been developed to describe H/D exchange in native proteins: the “solvent penetration” model (
      • Woodward C.
      • Simon I.
      • Tuchsen E.
      Hydrogen exchange and the dynamic structure of proteins..
      ) and the “local unfolding” model (
      • Englander S.W.
      • Englander J.J.
      • McKinnie R.E.
      • Ackers G.K.
      • Turner G.J.
      • Westrick J.A.
      • Gill S.J.
      Hydrogen exchange measurements of the free energy of structural and allosteric change in hemoglobin..
      ). According to the solvent penetration model, protons exchange within the protein interior as catalysts enter the protein core through transiently formed channels and cavities. Therefore, the rate of exchange of a proton depends on its average accessibility to solvent, which is a function of its depth and the local mobility of the protein’s structural elements, and on its reactivity with the catalyst, which is affected by local structural features, packing density, and hydrogen bonding. In the local unfolding model, exchange occurs on the surface of the polypeptide chain when a subregion transiently unfolds, exposing the surfaces to bulk solvent. In this model, the main barrier to exchange is considered to be the interior hydrogen bonding of amide protons, rather than their depth. Adjacent protons within subregions are predicted to exchange at roughly the same rate. It is generally accepted that the exchange rate of protein amide hydrogens is highly dependent on the combination of hydrogen bonding and the extent of shielding from solvent.
      The deuterium exchange rate in various segments of proteins can be semiquantitatively estimated from the time course of deuterium incorporation. Under conditions of constant pH, temperature, and bulk deuterated buffer, deuterium incorporation is described as the sum of a series of first-order exponentials of rate (
      • Englander S.W.
      • Kallenbach N.R.
      Hydrogen exchange and structural dynamics of proteins and nucleic acids..
      ):
      D=Ni=1Nexp(kit)
      (Eq. 6)


      where D is the deuterium content of a peptide with N peptide amide hydrogens as described for Eq. 1 and ki is the rate constant for exchange for the peptide hydrogens. Nonlinear least-squares fitting is used to obtain exchange rate constants for different groups of amide hydrogens from the MS data (
      • Bai Y.
      • Milne J.S.
      • Mayne L.
      • Englander S.W.
      Primary structure effects on peptide group hydrogen exchange..
      ).
      It is difficult to directly relate H/D exchange rates with protein structural features, though these may be revealed by judicious comparisons with calculated solvent accessibility in different segments of a protein or the conformational changes arising from environmental influences (
      • Buijs J.
      • Vera C.C.
      • Ayala E.
      • Steensma E.
      • Hakansson P.
      • Oscarsson S.
      Conformational stability of adsorbed insulin studied with mass spectrometry and hydrogen exchange..
      • Katta V.
      • Chait B.T.
      H/D exchange electrospray ionization mass spectrometry: a method for probing protein conformational changes in solution..
      • Thevenon-Emeric G.
      • Kozlowski J.
      • Zhang Z.
      • Smith D.L.
      Determination of amide hydrogen exchange rates in peptides by mass spectrometry..
      • Dharmasiri K.
      • Smith D.L.
      Mass spectrometric determination of isotopic exchange rates of amide hydrogens located on the surfaces of proteins..
      • Hughes C.A.
      • Mandell J.G.
      • Anand G.S.
      • Stock A.M.
      • Komives E.A.
      Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB..
      ) and on protein folding intermediates (
      • Miranker A.
      • Robinson C.V.
      • Radford S.E.
      • Aplin R.T.
      • Dobson C.M.
      Detection of transient protein folding populations by mass spectrometry..
      ,
      • Deng Y.
      • Smith D.L.
      Identification of unfolding domains in large proteins by their unfolding rates..
      ). Attempts have been made to find correlations between exchange rates and protein structural parameters, including most notably intramolecular hydrogen bonding and secondary structures (
      • Zhang Z.
      • Smith D.L.
      Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation..
      ), solvent accessibility surface areas (
      • Jeng M.-F.
      • Dyson H.J.
      Comparison of the hydrogen-exchange behavior of reduced and oxidized Escherichia coli thioredoxin..
      ), and B factors (
      • Hughes C.A.
      • Mandell J.G.
      • Anand G.S.
      • Stock A.M.
      • Komives E.A.
      Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB..
      ,
      • Zhang Z.
      • Post C.B.
      • Smith D.L.
      Amide hydrogen exchange determined by mass spectrometry: Application to rabbit muscle aldolase..
      ). Good correlations have been found for each in individual proteins. Such correlations were used to map three-dimensional topological details of the C terminus of rhM-CSFβ (
      • Yan X.
      • Zhang H.
      • Watson J.
      • Schimerlik M.I.
      • Deinzer M.L.
      H/D exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)..
      ), a protein that has not been crystallized.
      This homodimeric protein contains nine disulfide bonds. Proteins with multiple disulfide bonds present some major challenges in studies involving H/D exchange and MS analysis. Nevertheless, isotopic exchange results were obtained for rhM-CSFβ with a spatial resolution of 3–31 residues by simultaneously reducing the disulfide bonds and proteolytically digesting the native protein under quench conditions after exchange-in (
      • Yan X.
      • Zhang H.
      • Watson J.
      • Schimerlik M.I.
      • Deinzer M.L.
      H/D exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)..
      ). X-ray crystallographic coordinates of atoms in aa 4–149 are available for rhM-CSFα, whose structure is identical with the core of rhM-CSFβ. The latter has an extended C terminus consisting of aa 150–221. The crystallographic data allowed calculation of the average depth of residues and solvent-accessible surface area values. Deuterium exchange for all amide hydrogens in the long C-terminal tail, aa 150–221, was fast (kex > 4.16 min−1). It was clear that this segment was substantially solvent accessible and, therefore, believed to be largely unstructured in solution.
      The dead time (10 s) for deuterium exchange corresponds to an exchange rate constant, kex > 4.16 min−1. The amide hydrogen exchange rates within peptides for rhM-CSFβ were grouped into three categories, i.e. slow plus nonexchanging (kex < 0.1min−1), intermediate (0.1 min−1kex ≤ 4.16 min−1), and fast exchange (kex > 4.16 min−1). Residue depth was found to correlate with these hydrogen isotope exchange rates (105). Residues deeper than 6.0 Å have zero solvent accessibility, and the average calculated depth for the fast exchanging group in rhM-CSFβ is 4.8 Å (104). Thus, individual residues in the peptides were categorized into three groups; deep (>6.0 Å), intermediate (4.8 Å < average ≤ 6.0 Å), and shallow (≤4.8 Å).
      Generally, there was good correlation between the corresponding numbers of fast-exchanging amide hydrogens with shallow depths in 20 peptic fragments (Fig. 8 A). However, in the plot of the intermediate and slow plus nonexchanging groups, the peptides in the regions aa 95–105, aa 114–128, and aa 63–76 appear to be outliers (Fig. 8, B and C). The amide hydrogens in these peptides exchange too slowly relative to the depths calculated from the rhM-CSFα crystallographic data. The most obvious explanation for this discrepancy is that the long carboxyl-terminal tails, which are not present in rhM-CSFα, provide protective interactions with these segments. It may reasonably be concluded that the two C-terminal tails, which extend from Val152 in each subunit, meet at the center of the homodimer where two intermolecular disulfide bonds C155–C155′ and C157–C157′ link them together. Based on the crystal structure, the segment, aa 143–151, extends right below the region of the aa 95–105 segment, which includes the β2 strand, and provides some protection for it perhaps through induced hydrogen bonding. In addition, the C155–C155′ and C157–C157′ disulfide bonds in the native structure cause the C-terminal tails to extend across the surfaces of the aa 114–128 segment in αD and the aa 63–76 segment of αC on either side of the two domains, shielding them against amide H/D exchange (Fig. 9) (
      • Yan X.
      • Zhang H.
      • Watson J.
      • Schimerlik M.I.
      • Deinzer M.L.
      H/D exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)..
      ). Solvent-exposed surface area analysis showed the same results. The exposures of the amino acids, as calculated using the Connally method, were grouped into three categories: 1) highly exposed (≥50 Å2), 2) moderately exposed (30–50 Å2), and 3) buried (<30 Å2). The numbers of amino acids in peptides for each of these categories correlated with the corresponding numbers of residues in the three H/D exchange categories, and peptide aa 114–128 was again predicted to be significantly more accessible to solvent than the H/D exchange data suggested (
      • Yan X.
      • Zhang H.
      • Watson J.
      • Schimerlik M.I.
      • Deinzer M.L.
      H/D exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)..
      ).
      Figure thumbnail gr8
      Fig. 8Correlations of the number of amide hydrogens in (A) fast exchange, (B) intermediate exchange, and (C) slow plus nonexchange regimes in rhM-CSFβ with corresponding amide hydrogens at shallow, intermediate, and deep depths, respectively. The primary anomaly was seen in peptide aa 114–128, aa 63–76, and a second anomaly in aa 95–105. These segments are, therefore, protected from solvent to a greater extent than estimated on the basis of depth calculations from rhM-CSFα crystallographic data, which lead to their proposed interaction with the C-terminal tail.
      Figure thumbnail gr9
      Fig. 9Ribbon model of rhM-CSFβ. A, The structure of rhM-CSFβ up to Glu177 (of the full-length 218-residue protein) correlates better to H/D exchange data from MS than does the crystal structure of the truncated rhM-CSFα (truncated at His151). The rhM-CSFβ was constructed by first extending the crystal structure of rhM-CSFα to residue 160 (red). This allowed Cys157 to be cross-linked to Cys159 of the symmetry related dimer, and vice versa. The resulting structure was subjected to molecular dynamics and energy optimization using the AMBER force field. The remaining sequence to Glu177 was modeled as a turn and a polyproline type II helix (orange). This C-terminal helix buries the αD helix of the core protein (gray), which helps to rationalize the anomalously slow exchange seen for these residues when compared with the crystal structure. In addition, the disulfide cross-link tethers residues 151–160 across the dimer interface. This is consistent with the observation that H/D exchange at residues 95–105 (violet) is less than predicted by depth analysis from rhM-CSFα crystallographic data, and reduction of the disulfide bonds C157–C159 exposes more charge sites as observed by ESI-MS in rhM-CSFβ than was originally expected (
      • Zhang Y.H.
      • Yan X.
      • Maier C.S.
      • Schimerlik M.I.
      • Deinzer M.L.
      Structural comparison of recombinant human macrophage colony stimulating factor b and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry..
      ). B is the top view of the model.
      Modeling provided additional support for the proposed structure. A model was first constructed by extending the crystal structure of rhM-CSFα to 160, thereby providing the symmetrical C157–C159 disulfide linkages in the dimer. The resulting structure was subjected to molecular dynamics and energy optimization by AMBER force field and the remaining sequence to Glu177 was then modeled as a turn and a polyproline type II helix. In the resulting model (Fig. 9), the C-terminal helix buries the αD helix of the protein consistent with the experimental results. Moreover, the segments aa 95–105 lie beneath the C-terminal regions in the dimer that can otherwise become exposed when the C157–C159 disulfide linkages are reduced.
      An increase of 12 charges in the ESI spectrum when these two disulfide bonds are reduced can now be more fully appreciated (
      • Zhang Y.H.
      • Yan X.
      • Maier C.S.
      • Schimerlik M.I.
      • Deinzer M.L.
      Structural comparison of recombinant human macrophage colony stimulating factor b and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry..
      ). In the segment aa 95–105, there are three basic sites, His98, Lys100, and Arg104, which become exposed in the two subunits as a result of reduction of the disulfide bonds. Thus, three additional basic sites can be accounted for in each subunit for a total of six in the homodimer or eight if Lys154 is included. Previously, it was somewhat difficult to rationalize the 12 additional charges (
      • Zhang Y.H.
      • Yan X.
      • Maier C.S.
      • Schimerlik M.I.
      • Deinzer M.L.
      Structural comparison of recombinant human macrophage colony stimulating factor b and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry..
      ) as only Lys154 becomes exposed when the C157–C159 disulfides are reduced. These results would not have been available from x-ray studies because the full-length protein has not been crystallized.

      CONCLUSIONS AND PROSPECTS

      MS is becoming an increasingly important tool to study protein structures and dynamics. It provides complementary information for studying protein higher-order structure or conformational changes with other biophysical methods. It has a unique capacity to detect co-existing conformations and to monitor conformational changes in relatively fast processes (i.e. millisecond timescale) either by CSD in ESI-MS or in combination with H/D exchange techniques that can provide information on three-dimensional structures at 5- to 10-residue spatial resolution. Site-specific amide H/D exchange analyzed by CID MS/MS is potentially a very powerful approach for examining interactions at the individual residue level, if label scrambling can be eliminated and proteolytic digestion and incomplete collisional fragmentation can be improved upon. In addition, MS provides high sensitivity, relatively fast analysis, and a wide range of solvent, pH, and temperature conditions under which proteins and peptides can be examined. Protein size also is not limiting, and together with the inherent sensitivity of MS the risk of protein aggregation is reduced. Recent advances in mass resolving power and mass measurement accuracies on modern instruments will surely have an impact by removing any doubt about charge states and modifications that result in relatively small changes in mass, such as cyanylation, oxidation, methylation, etc. It is expected that in the future the structures of proteins that fail to crystallize may potentially be partially characterized by a combination of H/D exchange methods and judicious use of modeling, sequence-dependent secondary structure predictions, and comparisons with related or homologous proteins in databases. Future developments are likely also to involve electron capture dissociation on instruments that are accessible to the average user, thereby providing robustness in H/D analysis and structure determinations. Electron capture dissociation should reduce the potential for label scrambling and provide greater opportunities to perform structural analysis on whole proteins in the gas phase. Back-exchange attendant with current fragmentation methods may be minimized by conducting on-line H/D exchange, on-line fragmentation, and faster analyses, or by fragmenting intact proteins directly in the mass spectrometer to yield site-specific information. Automation in H/D analysis is a potentially powerful technique for rapid analysis of ligand, agonist, and antagonist binding to proteins in pharmaceutical chemistry and protein-protein and protein-DNA interactions for studies in signaling pathways.

      Acknowledgments

      The results from M. Kim and H. Zhang contributed in this review are gratefully acknowledged.

      REFERENCES

        • Clague M.J.
        Molecular aspects of the endocytic pathway..
        Biochem. J. 1998; 336: 271-282
        • Fink A.L.
        Compact intermediate states in protein folding..
        Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 495-522
        • Parkhomenko T.V.
        • Klicenko O.A.
        • Shavlovski M.M.
        • Kuznetsova I.M.
        • Uversky V.N.
        • Turoverov K.K.
        Biophysical characterization of albumin preparations from blood serum of healthy donors and patients with renal diseases. Part I: Spectrofluorometric analysis..
        Mei. Sci. Monitor. 2002; 8: 261-265
        • Pelton J.T.
        • McLean L.R.
        Spectroscopic methods for analysis of protein secondary structure..
        Anal. Biochem. 2000; 277: 167-176
        • Bucci E.
        • Steiner R.F.
        Anisotropy decay of fluorescence as an experimental approach to protein dynamics..
        Biophys. Chem. 1988; 30: 199-224
        • Barth A.
        • Zscherp C.
        Substrate binding and enzyme function investigated by infrared spectroscopy..
        FEBS Let. 2000; 477: 151-156
        • Ringe D.
        • Petsko G.A.
        Mapping protein dynamics by x-ray diffraction..
        Prog. Biophys. Mol. Biol. 1985; 45: 197-235
        • Ferentz A.E.
        • Wagner G.
        NMR spectroscopy: A multifaceted approach to macromolecular structure..
        Q. Rev. Biophys. 2000; 33: 29-65
        • Ohki S.-Y.
        • Eto M.
        • Kariya E.
        • Hayano T.
        • Hayashi Y.
        • Yazawa M.
        • Brautigan D.
        • Kainosho M.
        Solution NMR structure of the myosin phosphatase inhibitor protein CPI-17 shows phosphorylation-induced conformational changes responsible for activation..
        J. Mol. Biol. 2001; 314: 839-849
        • Hooke S.D.
        • Radford S.E.
        • Dobson C.M.
        The refolding of human lysozyme: A comparison with the structurally homologous hen lysozyme..
        Biochemistry. 1994; 33: 5867-5876
        • Tolman J.R.
        • Flanagan J.M.
        • Kennedy M.A.
        • Prestegard J.H.
        NMR evidence for slow collective motions in cyanometmyoglobin..
        Nat. Struct. Biol. 1997; 4: 292-297
        • Chowdhury S.K.
        • Katta V.
        • Chait B.T.
        Probing conformational changes in proteins by mass spectrometry..
        J. Am. Chem. Soc. 1990; 112: 9012-9013
        • Wang G.
        • Cole R.B.
        Electrospray Ionization Mass Spectrometry Fundamentals, Instrumentation, and Applications. Solution, Gas-phase, and Instrumental Parameter Influences on Charge-State Distributions in Electrospray Ionization Mass Spectrometry. John Wiley & Sons, Inc., New York1997: 137-174
        • Gumerov D.R.
        • Dobo A.
        • Kaltashov I.A.
        Protein-ion charge-state distributions in electrospray ionization mass spectrometry: Distinguishing conformational contributions from masking effects..
        Eur. J. Mass Spectrom. 2002; 8: 123-129
        • Mirza U.A.
        • Cohen S.L.
        • Chait B.T.
        Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry..
        Anal. Chem. 1993; 65: 1-6
        • Maier C.S.
        • Schimerlik M.I.
        • Deinzer M.L.
        Thermal denaturation of Escherichia coli thioredoxin studied by H/D exchange and electrospray ionization mass spectrometry: Monitoring a two-state protein unfolding transition..
        Biochemistry. 1999; 38: 1136-1143
        • Iavarone A.T.
        • Jurchen J.C.
        • Williams E.R.
        Effects of solvent on the maximum charge state and CSD of protein ions produced by electrospray ionization..
        J. Am. Soc. Mass Spectrom. 2000; 11: 976-985
        • Farmer T.B.
        • Caprioli R.M.
        Electrospray ionization mass spectrometry: Protein structure..
        NATO ASI Ser. C. 1996; 475: 61-88
        • Schnier P.D.
        • Gross D.S.
        • Williams E.R.
        On the maximum charge state and proton transfer reactivity of peptide and protein ions formed by electrospray ionization..
        J. Am. Soc. Mass Spectrom. 1995; 6: 1086-1097
        • Smith R.D.
        • Loo J.A.
        • Ogorzalek Loo R.R.
        • Busman M.
        • Udseth H.R.
        Principles and practice of electrospray ionization-mass spectrometry for large polypeptides and proteins..
        Mass Spectrom. Rev. 1991; 10: 359-452
        • Konermann L.
        • Douglas D.J.
        Acid-induced unfolding of cytochrome c at different methanol concentrations: Electrospray ionization mass spectrometry specifically monitors changes in the tertiary structure..
        Biochemistry. 1997; 36: 12296-12302
        • Loo J.A.
        • Edmonds C.G.
        • Udseth H.R.
        • Smith R.D.
        Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra..
        Anal. Chem. 1990; 62: 693-698
        • Nesatyy V.J.
        Gas-phase binding of non-covalent protein complexes between bovine pancreatic trypsin inhibitor and its target enzymes studied by electrospray ionization tandem mass spectrometry..
        J. Mass Spectrom. 2001; 36: 950-959
        • Wang F.
        • Freitas M.A.
        • Marshall A.G.
        • Sykes B.D.
        Gas-phase memory of solution-phase protein conformation: H/D exchange and Fourier transform ion cyclotron resonance mass spectrometry of the N-terminal domain of cardiac troponin C..
        Int. J. Mass Spectrom. 1997; 192: 319-325
        • Chillier X.F.D.
        • Monnier A.
        • Bill H.
        • Gülaçar F.O.
        • Buchs A.
        • McLuckey S.A.
        • Berkel G.J.V.
        A mass spectrometry and optical spectroscopy investigation of gas-phase ion formation in electrospray..
        Rapid Commun. Mass Spectrom. 1996; 10: 299-304
        • Loo J.A.
        • Loo R.R.O.
        Applying charge discrimination with electrospray ionization-mass spectrometry to protein analyses..
        J. Am. Soc. Mass Spectrom. 1995; 6: 1098-1104
        • Wang G.
        • Cole R.B.
        Mechanistic interpretation of the dependence of CSDs on analyte concentrations in electrospray ionization mass spectrometry..
        Anal. Chem. 1995; 67: 2892-2900
        • Mao D.
        • Babu K.R.
        • Chen Y.L.
        • Douglas D.J.
        Conformations of gas-phase lysozyme ions produced from two different solution conformations..
        Anal. Chem. 2003; 75: 1325-1330
        • Belva H.
        • Lange C.
        Conformational properties of a-dendrotoxin using electrospray mass spectrometry..
        Eur. J. Mass Spectrom. 2001; 7: 373-383
        • Sterner J.L.
        • Johnston M.V.
        • Nicol G.R.
        • Ridge D.P.
        Apparent proton affinities of highly charged peptide ions..
        J. Am. Soc. Mass Spectrom. 1998; 10: 483-491
        • Loo J.A.
        • He J.X.
        • Cody W.L.
        Higher order structure in the gas phase reflects solution structure..
        J. Am. Chem. Soc. 1998; 120: 4542-4543
        • Loo R.R.O.
        • Smith R.D.
        Investigation of the gas-phase structure of electrosprayed proteins using ion-molecule reactions..
        J. Am. Soc. Mass Spectrom. 1994; 5: 207-220
        • Carbeck J.D.
        • Severs J.C.
        • Gao J.
        • Wu Q.
        • Smith R.D.
        • Whitesides G.M.
        Correlation between the charge of proteins in solution and in the gas phase investigated by protein charge ladders, capillary electrophoresis, and electrospray ionization mass spectrometry..
        J. Phys. Chem. B. 1998; 102: 10596-10601
        • Covey T.R.
        • Bonner R.F.
        • Shushan B.I.
        • Henion J.J.
        The determination of protein, oligonucleotide and peptide molecular weights by ion-spray mass spectrometry..
        Rapid Commun. Mass Spectrom. 1988; 2: 249-256
        • Halgand F.
        • Laprevote O.
        Mean charge state and CSD of proteins as structural probes. An electrospray ionisation mass spectrometry study of lysozyme and ribonuclease A..
        Eur. J. Mass Spectrom. 2001; 7: 433-439
        • Fenn J.B.
        Ion formation from charged droplets: Roles of geometry, energy, and time..
        J. Am. Soc. Mass Spectrom. 1993; 4: 524-535
        • Kuloglu E.S.
        • McCaslin D.R.
        • Markley J.L.
        • Volkman B.F.
        Structural rearrangement of human lymphotactin, a C chemokine, under physiological solution conditions..
        J. Biol. Chem. 2002; 277: 17863-17870
        • Kaltashov I.A.
        • Eyles S.J.
        Studies of biomolecular conformations and conformational dynamics by mass spectrometry..
        Mass Spectrom. Rev. 2002; 21: 37-71
        • Dobo A.
        • Kaltashov I.A.
        Detection of multiple protein conformational ensembles in solution via deconvolution of charge-state distributions in ESI MS..
        Anal. Chem. 2001; 73: 4763-4773
        • Privalov P.L.
        Physical Basis of the Stability of the Folded Conformations of Proteins, W. H. Freeman and Co., New York1992
        • Creighton T.E.
        Protein folding..
        Biochem. J. 1990; 270: 1-16
        • Kim M.-Y.
        • Maier C.S.
        • Reed D.J.
        • Deinzer M.L.
        Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
        Protein Sci. 2002; 11: 1320-1329
        • Kim M.-Y.
        • Maier C.S.
        • Reed D.J.
        • Ho P.S.
        • Deinzer M.L.
        Intramolecular interactions in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry..
        Biochemistry. 2001; 40: 14413-14421
        • Bremer E.T.J.V.D.
        • Jiskoot W.
        • James R.
        • Moore G.R.
        • Kleanthous C.
        • Heck A.J.R.
        • Maier C.S.
        Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry..
        Protein Sci. 2002; 11: 1738-1752
        • Zhang Y.H.
        • Yan X.
        • Maier C.S.
        • Schimerlik M.I.
        • Deinzer M.L.
        Structural comparison of recombinant human macrophage colony stimulating factor b and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry..
        Protein Sci. 2001; 10: 2336-2345
        • Wagner D.S.
        • Anderegg R.J.
        Conformation of cytochrome c studied by deuterium exchange-electrospray ionization mass spectrometry..
        Anal. Chem. 1994; 66: 706-711
        • Babu K.R.
        • Moradian A.
        • Douglas D.J.
        Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry..
        J. Am. Soc. Mass Spectrom. 2001; 12: 317-328
        • Eyles S.J.
        • Dresch T.
        • Gierasch L.M.
        • Kaltashov I.A.
        Unfolding dynamics of a beta-sheet protein studied by mass spectrometry..
        J. Mass Spectrom. 1999; 34: 1289-1295
        • Miranker A.
        • Robinson C.V.
        • Radford S.E.
        • Dobson C.M.
        Investigation of protein folding by mass spectrometry..
        FASEB J. 1996; 10: 93-101
        • Hvidt A.
        • Linderstrom-Lang K.
        Exchange of hydrogen atoms in insulin with deuterium atoms in aqueous solutions..
        Biochem. Biophys. Acta. 1954; 14: 574-575
        • Rosa J.J.
        • Richards F.M.
        An experimental procedure for increasing the structural resolution of chemical hydrogen-exchange measurements on proteins: Application to ribonuclease S peptide..
        J. Mol. Biol. 1979; 133: 399-416
        • Englander J.J.
        • Rogero J.R.
        • Englander S.W.
        Protein hydrogen exchange studied by the fragment separation method..
        Anal. Biochem. 1985; 147: 234-244
        • Katta V.
        • Chait B.T.
        Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry..
        Rapid Commun. Mass Spectrom. 1991; 5: 214-217
        • Eigen M.
        Proton transfer, acid-base catalysis, and enzymic hydrolysis..
        Angew. Chem. 1963; 75: 489-508
        • Bai Y.
        • Milne J.S.
        • Mayne L.
        • Englander S.W.
        Primary structure effects on peptide group hydrogen exchange..
        Proteins Struct. Funct. Genet. 1993; 17: 75-86
        • Smith D.L.
        • Deng Y.
        • Zhang Z.
        Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry..
        J. Mass Spectrom. 1997; 32: 135-146
        • Freitas M.A.
        • Hendrickson C.L.
        • Emmett M.R.
        • Marshall A.G.
        Gas-phase bovine ubiquitin cation conformations resolved by gas-phase H/D exchange rate and extent..
        Int. J. Mass Spectrom. 1999; 185–187: 565-575
        • Campbell S.
        • Rodgers M.T.
        • Marzluff E.M.
        • Beauchamp J.L.
        Deuterium exchange reactions as a probe of biomolecule structure. Fundamental studies of gas phase H/D exchange reactions of protonated glycine oligomers with D2O, CD3OD, CD3CO2D, and ND3..
        J. Am. Chem. Soc. 1995; 117: 12840-12854
        • Wyttenbach T.
        • Bowers M.T.
        Pressure limited sustained off-resonance irradiation for collision-activated dissociation in Fourier transform mass spectrometry..
        J. Am. Soc. Mass Spectrom. 1998; 10: 9-14
        • Freitas M.A.
        • Hendrickson C.L.
        • Emmett M.R.
        • Marshall A.G.
        Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry..
        J. Am. Soc. Mass Spectrom. 1998; 9: 1012-1019
        • Englander J.J.
        • Englander S.W.
        • Louie G.
        • Roder H.
        • Tran T.
        • Wand A.J.
        Protein Hydrogen Exchange, Dynamics, and Energetics. Adenine Press, Schenectady, NY1988: 107-117
        • Miranker A.
        • Robinson C.V.
        • Radford S.E.
        • Aplin R.T.
        • Dobson C.M.
        Detection of transient protein folding populations by mass spectrometry..
        Science. 1993; 262: 896-900
        • Matagne A.
        • Jamin M.
        • Chung E.W.
        • Robinson C.V.
        • Radford S.E.
        • Dobson C.M.
        Thermal unfolding of an intermediate is associated with non-arrhenius kinetics in the folding of hen lysozyme..
        J. Mol. Biol. 2000; 297: 193-210
        • Eyles S.J.
        • Radford S.E.
        • Robinson C.V.
        • Dobson C.M.
        Kinetic consequences of the removal of a disulfide bridge on the folding of hen lysozyme..
        Biochemistry. 1994; 33: 13038-13048
        • Zhang Z.
        • Smith D.L.
        Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation..
        Protein Sci. 1993; 2: 522-531
        • McLafferty F.W.
        • Guan Z.
        • Haupts U.
        • Wood T.D.
        • Kelleher N.L.
        Gaseous conformational structures of cytochrome c..
        J. Am. Chem. Soc. 1998; 120: 4732-4740
        • Demmers J.A.A.
        • Haverkamp J.
        • Heck A.J.
        • Koeppe R.E.
        • Killian J.A.
        Electrospray ionization mass spectrometry as a tool to analyze H/D exchange kinetics of transmembrane peptides in lipid bilayers..
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3189-3194
        • Demmers J.A.A.
        • Duijn E.v.
        • Haverkamp J.
        • Greathouse D.V.
        • Roger E. Koeppe I.
        • Heck A.J.R.
        • Killian J.A.
        Interfacial positioning and stability of transmembrane peptides in lipid bilayers studied by combining H/D exchange and mass spectrometry..
        J. Biol. Chem. 2001; 276: 34501-34508
        • Demmers J.A.A.
        • Rijkers D.T.S.
        • Haverkamp J.
        • Killian J.A.
        • Heck A.J.R.
        Factors affecting gas-phase deuterium scrambling in peptide ions and their implications for protein structure determination..
        J. Am. Chem. Soc. 2002; 124: 11191-11198
        • Jorgensen T.J.D.
        • Ploug M.
        • Roepstorff P.
        Mapping the binding interface between urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) by ESI-MS and amide H/D exchange, Proceedings of the 51th ASMS Conference, Montreal, Quebec, Canada2003
        • Schlosser A.
        • Lehmann W.
        Five-membered ring formation in unimolecular reactions of peptides: A key structural element controlling low-energy collision-induced dissociation of peptides..
        J. Mass Spectrom. 2000; 35: 1382-1390
        • Harrison A.G.
        • Siu K.W.M.
        • Aribi H.E.
        Amide bond cleavage in deprotonated tripeptides: A newly discovered pathway to “b2 ions..
        Rapid Commun. Mass Spectrom. 2003; 17: 869-875
        • Harrison A.G.
        • Yalcin T.
        Proton mobility in protonated amino acids and peptides..
        Int. J. Mass Spectrom. Ion Processes. 1997; 165/166: 339-347
        • Dongre A.R.
        • Jones J.L.
        • Somogyi A.
        • Wysocki V.H.
        Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: Evidence for the mobile proton model..
        J. Am. Chem. Soc. 2000; 118: 8365-8374
        • Eyles S.J.
        • Speir J.P.
        • Kruppa G.H.
        • Gierasch L.M.
        • Kaltashov I.A.
        Protein conformational stability probed by Fourier transform ion cyclotron resonance mass spectrometry..
        J. Am. Chem. Soc. 2000; 122: 495-500
        • Voyksner R.D.
        • Lee H.
        Investigating the use of an octupole ion guide for ion storage and high-pass mass filtering to improve the quantitative performance of electrospray ion trap mass spectrometry..
        Rapid Commun. Mass Spectrom. 1999; 13: 1427-1437
        • Kaltashov I.A.
        • Eyles S.J.
        Crossing the phase boundary to study protein dynamics and function: Combination of amide hydrogen exchange in solution and ion fragmentation in the gas phase..
        J. Mass Spectrom. 2002; 37: 557-565
        • Kim M.Y.
        • Maier C.S.
        • Reed D.J.
        • Deinzer M.L.
        Site-specific amide H/D exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry..
        J. Am. Chem. Soc. 2001; 123: 9860-9866
        • Deng Y.
        • Pan H.
        • Smith D.L.
        Selective isotope labeling demonstrates that hydrogen exchange at individual peptide amide linkages can be determined by collision-induced dissociation mass spectrometry..
        J. Am. Chem. Soc. 1999; 121: 1966-1967
        • Yan X.
        • Broderick D.
        • Leid M.E.
        • Schimerlik M.I.
        • Deinzer M.L.
        Dynamics and ligand-induced solvent accessibility changes in human retinoid X receptor homodimer determined by hydrogen deuterium exchange and mass spectrometry.
        Biochemistry,. 2003; (in press)
        • Alkashi S.
        • Naito Y.
        • Takio K.
        Observation of hydrogen-deuterium exchange of ubiquitin by direct analysis of electrospray capillary-skimmer dissociation with Fourier transform ion cyclotron resonance mass spectrometry..
        Anal. Chem. 1999; 71: 4974-4980
        • Hvidt A.
        • Nielsen S.O.
        Hydrogen exchange in proteins..
        Adv. Portein Sci. 1966; 21: 287-386
        • Englander J.J.
        • Calhoun D.B.
        • Englander S.W.
        Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry..
        Anal. Biochem. 1979; 92: 517-524
        • Woodward C.
        • Simon I.
        • Tuchsen E.
        Hydrogen exchange and the dynamic structure of proteins..
        Mol. Cell Biochem. 1982; 48: 135-160
        • Englander S.W.
        • Mayne L.
        Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR..
        Annu. Rev. Biophys. Biomol. Struct. 1992; 21: 243-265
        • Clarke J.
        • Itzhaki L.S.
        Hydrogen exchange and protein folding..
        Curr. Opin. Struct. Biol. 1998; 8: 112-118
        • Raschke T.
        • Marqusee S.
        Hydrogen exchange studies of protein structure..
        Curr. Opin. Biotech. 1998; 9: 80-86
        • Bahar I.
        • Wallqvist A.
        • Covell D.G.
        • Jernigan R.L.
        Correlation between native-state hydrogen and cooperative residue fluctuations from a simple model..
        Biochemistry. 1998; 37: 1067-1075
        • Bai Y.
        • Milne J.S.
        • Mayne L.
        • Englander S.W.
        Protein stability parameters measured by hydrogen exchange..
        Protein Struct. Funct. Genet. 1994; 20: 4-14
      1. Kim, M.-Y.(2000)Mass Spectrometric Studies on Peptides and Proteins: Conformations of Escherichia coli Thioredoxin and its Alkylated Adducts Studied by H/D Exchange and HPLC-Electrospray Ionization Mass Spectrometry. Ph. D. thesis, Oregon State University

        • Canet D.
        • Last A.M.
        • Tito P.
        • Sunde M.
        • Spencer A.
        • Archer D.B.
        • Redfield C.
        • Robinson C.V.
        • Dobson C.M.
        Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme..
        Nat. Struct. Biol. 2002; 9: 308-315
        • Englander S.W.
        • Englander J.J.
        • McKinnie R.E.
        • Ackers G.K.
        • Turner G.J.
        • Westrick J.A.
        • Gill S.J.
        Hydrogen exchange measurements of the free energy of structural and allosteric change in hemoglobin..
        Science. 1992; 256: 1684-1687
        • Englander S.W.
        • Kallenbach N.R.
        Hydrogen exchange and structural dynamics of proteins and nucleic acids..
        Q. Rev. Biophys. 1983; 16: 521-655
        • Buijs J.
        • Vera C.C.
        • Ayala E.
        • Steensma E.
        • Hakansson P.
        • Oscarsson S.
        Conformational stability of adsorbed insulin studied with mass spectrometry and hydrogen exchange..
        Anal. Chem. 1999; 71: 3219-3225
        • Katta V.
        • Chait B.T.
        H/D exchange electrospray ionization mass spectrometry: a method for probing protein conformational changes in solution..
        J. Am. Chem. Soc. 1993; 115: 6317-6321
        • Thevenon-Emeric G.
        • Kozlowski J.
        • Zhang Z.
        • Smith D.L.
        Determination of amide hydrogen exchange rates in peptides by mass spectrometry..
        Anal. Chem. 1992; 64: 2456-2458
        • Dharmasiri K.
        • Smith D.L.
        Mass spectrometric determination of isotopic exchange rates of amide hydrogens located on the surfaces of proteins..
        Anal. Chem. 1996; 68: 2340-2344
        • Hughes C.A.
        • Mandell J.G.
        • Anand G.S.
        • Stock A.M.
        • Komives E.A.
        Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB..
        J. Mol. Biol. 2001; 307: 967-976
        • Deng Y.
        • Smith D.L.
        Identification of unfolding domains in large proteins by their unfolding rates..
        Biochemistry. 1998; 37: 6256-6262
        • Jeng M.-F.
        • Dyson H.J.
        Comparison of the hydrogen-exchange behavior of reduced and oxidized Escherichia coli thioredoxin..
        Biochemistry. 1995; 34: 611-619
        • Zhang Z.
        • Post C.B.
        • Smith D.L.
        Amide hydrogen exchange determined by mass spectrometry: Application to rabbit muscle aldolase..
        Biochemistry. 1996; 35: 779-791
        • Yan X.
        • Zhang H.
        • Watson J.
        • Schimerlik M.I.
        • Deinzer M.L.
        H/D exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFβ)..
        Protein Sci. 2002; 11: 2113-2124