Regional stability changes in oxidized and reduced cytochrome c located by hydrogen exchange and mass spectrometry

Amide hydrogen exchange rates are highly sensitive to protein structure and may, therefore, be used to detect and characterize structural changes in proteins. Specific regions within folded proteins undergoing structural change can often be identified if localized amide hydrogen exchange rates are determined by nuclear magnetic resonance (NMR). The ability to measure localized amide hydrogen exchange rates by proteolytic fragmentation followed by mass spectrometric analysis opens the possibility to also identify localized structural changes in proteins by mass spectrometry. If successful, this approach offers considerable advantage over NMR in speed, sensitivity, protein solubility, and ability to study large proteins. This possibility has been investigated by determining the amide hydrogen exchange rates in oxidized and reduced cytochrome c by protein fragmentation/mass spectrometry. The fundamental difference in these forms of cytochrome c is the oxidation state of the iron, which other studies have shown results in only minor structural changes in the protein. In the present study, the largest differences in hydrogen exchange rates were found for peptide amide hydrogens located distant from the Nand C-termini, indicating that the structure in these regions is most affected by the oxidation state of the iron. These results are consistent with previous studies of oxidized and reduced cytochrome c, suggesting that hydrogen exchange and mass spectrometry may be generally useful for locating subtle changes in protein structure.     

Solvent accessibility of protein surfaces by amide H/2H exchange MALDI-TOF mass spectrometry

One advantage of detecting amide H/2H exchange by mass spectrometry instead of NMR is that the more rapidly exchanging surface amides are still detectable. In this study, we present quench-flow amide H/2H exchange experiments to probe how rapidly the surfaces of two different proteins exchange. We compared the amide H/2H exchange behavior of thrombin, a globular protein, and IkappaBalpha, a nonglobular protein, to explore any differences in the determinants of amide H/2H exchange rates for each class of protein. The rates of exchange of only a few of the surface amides were as rapid as the "intrinsic" exchange rates measured for amides in unstructured peptides. Most of the surface amides exchanged at a slower rate, despite the fact that they were not seen to be hydrogen bonded to another protein group in the crystal structure. To elucidate the influence of the surface environment on amide H/2H exchange, we compared exchange data with the number of amides participating in hydrogen bonds with other protein groups and with the solvent accessible surface area. The best correlation with amide H/2H exchange was found with the total solvent accessible surface area, including side chains. In the case of the globular protein, the correlation was modest, whereas it was well correlated for the nonglobular protein. The nonglobular protein also showed a correlation between amide exchange and hydrogen bonding. These data suggest that other factors, such as complex dynamic behavior and surface burial, may alter the expected exchange rates in globular proteins more than in nonglobular proteins where all of the residues are near the surface.     

Improved Sequence Resolution by Global Analysis of Overlapped Peptides in Hydrogen/Deuterium Exchange Mass Spectrometry

Management of the enormous amount of data produced during solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has stimulated software analysis development. The proteolysis step of the experiment generates multiple peptide fragments, most of which overlap. Prior automated data reduction algorithms extract the deuteration level for individual peptides, but do not exploit the additional information arising from fragment overlap. Here, we describe an algorithm that determines discrete rate constant values to each of the amide hydrogens in overlapped fragments. By considering all of the overlapped peptide segments simultaneously, sequence resolution can be improved significantly, sometimes to the individual amino acid level. We have validated the method with simulated deuterium uptake data for seven overlapped fragments of a poly-Ala nonapeptide, and then applied it to extract rate constant values for the first 29?N-terminal amino acids of C22A FK506-binding protein.     

Many Overlapping Peptides for Protein Hydrogen Exchange Experiments by the Fragment Separation-Mass Spectrometry Method

Measurement of the naturally occurring hydrogen exchange (HX) behavior of proteins can in principle provide highly resolved thermodynamic and kinetic information on protein structure, dynamics, and interactions. The HX fragment separation-mass spectrometry method (HX-MS) is able to measure hydrogen exchange in biologically important protein systems that are not accessible to NMR methods. In order to achieve high structural resolution in HX-MS experiments, it will be necessary to obtain many sequentially overlapping peptide fragments and be able to identify and analyze them efficiently and accurately by mass spectrometry. This paper describes operations which, when applied to four different proteins ranging in size from 140 to 908 residues, routinely provides hundreds of useful unique peptides, covering the entire protein length many times over. Coverage in terms of the average number of peptide fragments that span each amino acid exceeds 10. The ability to achieve these results required the integrated application of experimental methods that are described here and a computer analysis program, called ExMS, described in a following paper.     

Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: Plasmepsin 2 but not plasmepsin 4 is active under quenching conditions

Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0°C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies.     

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Direct mass spectrometric characterization of interactions between proteins and small hydrophobic ligands often poses a serious problem due to the complex instability in the gas phase. We have developed a method that probes the efficacy of ligand-protein interactions indirectly by monitoring changes in protein flexibility. The latter is assessed quantitatively using a combination of charge state distribution analysis and amide hydrogen exchange under both native and mildly denaturing conditions. The method was used to evaluate binding of a model protein cellular retinoic acid binding protein I to its natural ligand all-trans retinoic acid (RA), isomers 13-cis- and 9-cis-RA, and retinol, yielding the following order of ligand affinities: All-trans RA > 9-cis RA > 13-cis RA, with no detectable binding of retinol. This order is in agreement with the results of earlier fluorimetric titration studies. Furthermore, binding energy of the protein to each of retinoic acid isomers was determined based on the measured hydrogen exchange kinetics data acquired under native conditions.     

Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10–30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1–3 h). Here various experimental conditions, as well as a quenched‐flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044–1 000 000 s), when converted into the standard exchange condition (pH 7 and 23°C). Copyright © 2010 John Wiley & Sons, Ltd.     

Probing high order structure of proteins by fast-atom bombardment mass spectrometry

During the past decade, numerous investigations have demonstrated that the rate at which amide hydrogens located at peptide linkages undergo isotopic exchange is a sensitive probe of the high order structure and dynamics of proteins. The present investigation demonstrates that microbore high-performance liquid chromatography (HPLC) continuous-flow fast-atom bombardment mass spectrometry (FABMS) can be used to accurately quantify deuterium located at peptide linkages in short segments of large proteins. This result is important because it demonstrates the feasibility of using mass spectrometry as a tool for studying the high order structure and dynamics of large proteins. Following a period of deuterium exchange-in, a protein was placed into slow-exchange conditions and fragmented into peptides with pepsin. The digest was analyzed by continuous-flow HPLC FABMS to determine the molecular weights of the peptides, from which the number of deuterons located at the peptide linkages could be deduced. The HPLC step was used both to fractionate the peptides according to their hydrophobicities and to remove through back-exchange all deuterium except that located at peptide amide linkages. This approach has been applied to α-crystallin, a lens protein composed of two gene products with monomer molecular weights of 20 kDa and an aggregate molecular weight approaching 1000 kDa. Results from this study show that some of the peptide amide hydrogens in αA-crystallin exchange very rapidly (k > 10 h^?1) while others exchange very slowly (k < 10^?3 h^?1). The ability not only to detect that a conformational change has occurred, but also to identify the specific regions within the protein where the change occurred, was demonstrated by measuring changes in the exchange rates within these regions as the deuterium exchange-in temperature was increased from 10 to 80 ° C.     

Quantum mechanical calculations on dopamine D2-receptor antagonists: Conformation of remoxipride, eticlopride and NCQ 115

The quantum mechanical PCILO method has been used to study the conformation of three selective dopamine D2-receptor antagonists: remoxipride, eticlopride and NCQ 115. The calculations were done for both the protonated and ‘free base’ forms of the antagonists. The protonated antagonists all have folded structures due to intramolecular hydrogen bonding between the carbonyl of the amide moiety and the hydrogen on the pyrrolidine nitrogen. The ‘free bases’ on the other hand are characterised by extended structures. An interaction model for these molecules with the dopamine receptor has been proposed. This paper was presented at the Academy Discussion Meeting on “Recent trends in molecular structure and dynamics calculations” held at the Indian Institute of Technology, Kharagpur between February 5 and 7, 1993     

A new method for the accurate determination of the isotopic state of single amide hydrogens within peptides using Fourier transform ion cyclotron resonance mass spectrometry

A new method is presented to accurately determine the probability of having a deuterium or hydrogen atom on a specific amide position within a peptide after deuterium/hydrogen (D/H) exchange in solution. Amide hydrogen exchange has been proven to be a sensitive probe for studying protein structures and structural dynamics. At the same time, mass spectrometry in combination with physical fragmentation methods is commonly used to sequence proteins based on an amino acid residue specific mass analysis. In the present study it is demonstrated that the isotopic patterns of a series of peptide fragment ions obtained with capillary-skimmer dissociation, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, can be used to calculate the isotopic state of specific amide hydrogens. This calculation is based on the experimentally observed isotopic patterns of two consecutive fragments and on the isotopic binomial distributions of the atoms in the residue constituting the difference between these two consecutive fragments. The applicability of the method is demonstrated by following the sequence-specific D/H exchange rate in solution of single amide hydrogens within some peptides.     

Software Lock Mass by Two-Dimensional Minimization of Peptide Mass Errors

Mass accuracy is a key parameter in proteomic experiments, improving specificity, and success rates of peptide identification. Advances in instrumentation now make it possible to routinely obtain high resolution data in proteomic experiments. To compensate for drifts in instrument calibration, a compound of known mass is often employed. This ‘lock mass’ provides an internal mass standard in every spectrum. Here we take advantage of the complexity of typical peptide mixtures in proteomics to eliminate the requirement for a physical lock mass. We find that mass scale drift is primarily a function of the m/z and the elution time dimensions. Using a subset of high confidence peptide identifications from a first pass database search, which effectively substitute for the lock mass, we set up a global mathematical minimization problem. We perform a simultaneous fit in two dimensions using a function whose parameterization is automatically adjusted to the complexity of the analyzed peptide mixture. Mass deviation of the high confidence peptides from their calculated values is then minimized globally as a function of both m/z value and elution time. The resulting recalibration function performs equal or better than adding a lock mass from laboratory air to LTQ-Orbitrap spectra. This ‘software lock mass’ drastically improves mass accuracy compared with mass measurement without lock mass (up to 10-fold), with none of the experimental cost of a physical lock mass, and it integrated into the freely available MaxQuant analysis pipeline ().     

Hydrogen/deuterium exchange on yeast ATPase supramolecular protein complex analyzed at high sensitivity by MALDI mass spectrometry

To evaluate the ability of hydrogen/deuterium exchange of amide protons followed by mass spectrometry (HXMS) to yield topological information about supramolecular protein complexes, this approach has been tested with the 370 kDa hetero-oligomeric complex of yeast F1-ATPase. The study was focused on the epsilon subunit (6612 Da) of the complex. Deuterium back exchange due to the chromatographic isolation step of this subunit was strongly reduced by means of fast micro-chromatography, and MALDI-MS was used to analyze either the intact subunit or peptide mixtures resulting from its proteolytic cleavage. A deuterium labeling kinetic study was conducted with epsilon subunit being a part of the F1 native complex. The effect of a secondary structure was also investigated by means of HXMS on the isolated epsilon subunit. Finally, to determine which regions of epsilon subunit are accessible to solvent in F1-ATPase during exchange, the complex was submitted to hydrogen/deuterium exchange, the epsilon subunit was purified by micro-chromatography, digested by pepsin, and resulting peptide fragments were analyzed by MALDI-MS. The combination of hydrogen/deuterium exchange, fast micro-chromatography and MALDI-MS was shown to be a fast and efficient way to obtain detailed topological information for the epsilon subunit when it is engaged in the ATPase complex.     

Is there hydrogen scrambling in the gas phase? Energetic and structural determinants of proton mobility within protein ions

The extent of internal hydrogen exchange (scrambling) within multiply charged solvent-free protein ions was investigated using a small model protein. The site-specific backbone amide protection data were obtained using protein ion fragmentation in the gas phase and compared with the available NMR data. Only minimal scrambling was detected when relatively high-energy collisional activation was used to fragment intact protein ions, while low-energy fragmentation resulted in more significant but not random internal exchange. Increased conformational flexibility of protein ions in the gas phase did not have any effect on the extent of hydrogen scrambling under the conditions of higher-energy collisional activation but resulted in totally random redistribution of labile hydrogen atoms when the protein ion fragmentation was induced by multiple low-energy collisions.     

IR spectra of N-methylacetamide in water predicted by combined quantum mechanical/molecular mechanical molecular dynamics simulations

We applied the combined quantum mechanical (QM)/molecular mechanical (MM) molecular dynamics (MD) simulation method in assessing IR spectra of N-methylacetamide and its deuterated form in aqueous solutions. The model peptide is treated at the Austin Model 1 (AM1) level and the induced dipole effects by the solvent are incorporated in fluctuating solute dipole moments, which are calculated using partial charges from Mulliken population analyses without resorting to any available high-level ab initio dipole moment data. Fourier transform of the solute dipole autocorrelation function produces in silico IR spectra, in which the relative peak intensities and bandwidths of major amide bands are quantitatively compatible with experimental results only when both geometric and electronic polarizations of the peptide by the solvent are dealt with at the same quantum-mechanical level. We cast light on the importance of addressing dynamic charge fluctuations of the solute in calculating IR spectra by comparing classical and QM/MM MD simulation results. We propose the adjustable scaling factors for each amide mode to be directly compared with experimental data.     

Hydrogen exchange mass spectrometry as an analytical tool for the analysis of amyloid fibrillogenesis

In this study, we have used glucagon as a model system for analyzing amyloid fibrillogenesis by hydrogen exchange MALDI mass spectrometry (HXMS). The hydrogen exchange mass spectrometry data correlated well with the traditional method based on Thioflavin T fluorescence and provided quantitative information by measuring the fibrillating molecules directly. The hydrogen exchange mass spectrometry data collected during fibrillogenesis revealed that glucagon fibrillation was a two component system showing an on/off type of interaction where only monomeric and fibrils were present without any substantial amount of intermediate species. This was evident by the extensive deuteration of the monomer and protection of the entire 29 residue glucagon peptide upon fibrillation.. The method complements the traditional procedures and has the potential to provide new information with respect to the nature of transient species, the structure of the growing fibrils and the mechanism of formation.     

Site-specific amide hydrogen exchange in melittin probed by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

Electron capture dissociation (ECD) has been proposed to be a non-ergodic process, i.e. to provide backbone dissociation of gas-phase peptides faster than randomization of the imparted energy. One potential consequence could be that ECD can fragment deuterated peptides without causing hydrogen scrambling and thereby provide amino acid residue-specific amide hydrogen exchange rates. Such a feature would improve the resolution of approaches involving solution-phase amide hydrogen exchange combined with mass spectrometry for protein structural characterization. Here, we explore this hypothesis using melittin, a haemolytic polypeptide from bee venom, as our model system. Exchange rates in methanol calculated from consecutive c-type ion pairs show some correlation with previous NMR data: the amide hydrogens of leucine 13 and alanine 15, located at the unstructured kink surrounding proline 14 in the melittin structure adopted in methanol, appear as fast exchangers and the amide hydrogens of leucine 16 and lysine 23, buried within the helical regions of melittin, appear as slow exchangers. However, calculations based on c-type ions for other amide hydrogens do not correlate well with NMR data, and evidence for deuterium scrambling in ECD was obtained from z*-type ions.     

Protein-peptide affinity determination using an H/D exchange dilution strategy: Application to antigen-antibody interactions

A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX) [J. Am. Chem. Soc. 2003, 125, 5252–5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide β-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques.     

Characterization of peptide-pyrazole interactions in solution by low-temperature NMR studies

Complexation of the amino- and carboxyl-protected tripeptide Piv-L-Val-L-Val-L-Val-tBu with 3-methylpyrazole and 3-amino-5-methylpyrazole was studied by low-temperature NMR experiments in a freonic solvent. The peptide forms an extended beta-type structure at all temperatures and associates through hydrogen bonding with the two pyrazole-based beta-sheet ligands. A detailed structural characterization of the formed complexes by one- and two-dimensional NMR experiments under slow exchange conditions was made possible by employing very low temperatures. The tripeptide associates to stable antiparallel dimers that are symmetrically capped on both sides by two pyrazole receptors to form 2:2 complexes. Amide groups of two neighboring residues in an extended conformation are involved in cyclic hydrogen bonds to the pyrazole. Based on amide chemical shift changes, the relative strength of intermolecular hydrogen bonds can be assessed and correlated with the electronic effects of the substituents on the pyrazole.     

The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange

We describe the use of H/D amide exchange and electrospray ionization mass spectrometry to study, in organic solvents, the pentadecapeptide gramicidin as a model for protein self association. In methanol-OD, all active H’s in the peptide exchange for D within 5 min, indicating a monomer/dimer equilibrium that is shifted towards the fast-exchanging monomer. H/D exchange in n-propanol-OD, however, showed a partially protected gramicidin that slowly converts to a second species that exchanges nearly all the active hydrogens, indicating EX1 kinetics for the H/D exchange. We propose that this behavior is the result of the slower rate of unfolding in n-propanol compared with that in methanol. The rate constant for the unfolding of the dimer is the rate of disappearance of the partially protected species, and it agrees within a factor of two with a value reported in literature. The rate constant of dimer refolding can be determined from the ratio of the rate constant for unfolding and the affinity constant for the dimer, which we determined in an earlier study. The unfolding activation energy is 20 kcal mol⁻¹, determined by performing the exchange experiments as a function of temperature. To study gramicidin in an even more hydrophobic medium than n-propanol, we measured its H/D exchange kinetics in a phospholipids vesicle and found a different H/D amide exchange behavior. Gramicidin is an unusual peptide dimer that can exhibit both EX1 and EX2 mechanisms for its H/D exchange, depending on the solvent.     

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and hydrogen exchange combined with enzymatic digestion for the structural characterization of antimalaric Spf66 peptide

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used in hydrogen exchange studies, exchanging deuteron (H/D) or proton (D/H), to determine the structure and conformational changes of antimalarial Spf66 synthetic peptide in its monomeric and dimeric forms. The accuracy of both analytical methods was assessed along with their suitability to study structural aspects. The results via these two approaches were in agreement, indicating that the dimer presents segments of secondary structure. In this last case, the combination of both methods with enzymatic digestion with pepsin was used in their identification. Although 100% coverage of Spf66 dimer was not observed, the higher levels of deuteration were observed for fragments located in the chain terminal where the structure may be more flexible, while the fragments near the disulfide bonds, which is, in theory, the more rigid region of the molecule, were not detected. This strategy is significantly time saving and allows rapid screening and help to characterize a protein, especially, when no prior structural information is available. However, a single spectrum is not certainly sufficient to obtain structural data; it is just an experimental limitation.Also, changes in peptide structure after storage at different temperatures and time were observed, which lead to a loss in the secondary structure as determined by circular dicroism measurements and an increase in aggregation products, since the trimer and tetramer species were detected by mass spectrometry.