Studying protein-carbohydrate interactions by amide hydrogen/deuterium exchange mass spectrometry

Protein-carbohydrate interactions play a significant role in biological processes. Presented here is the novel application of amide hydrogen/deuterium exchange mass spectrometry (amide exchange-MS) to the study of the interaction between a protein and its carbohydrate substrate. The degree of deuterium incorporation into hen egg lysozyme was monitored with and without substrate to verify that a carbohydrate can provide sufficiently stable protection of the amide hydrogen atoms in a protein's backbone from exchange with deuterated solvent. The substrate protected a number of amide hydrogens from exchange, implying that protein-carbohydrate binding systems will be compatible with amide exchange-MS. Endopolygalacturonase-II (EPG-II) from Aspergillus niger, a pectin-degrading enzyme, was chosen as the first carbohydrate-binding system to be extensively studied using quenched amide exchange-MS. Monitoring the changes in deuterium incorporation of EPG-II in the presence and absence of an oligomer of galacturonic acid implied the location of substrate binding. This study demonstrates the ability of amide exchange-MS to investigate protein-carbohydrate interactions.     

Fourier transform mass spectrometry to monitor hyaluronan-protein interactions: use of hydrogen/deuterium amide exchange

The use of Fourier transform mass spectrometry (FTMS) to monitor noncovalent complex formation in the gas phase under native conditions between the Link module from human tumor necrosis factor stimulated gene-6 (Link_TSG6) and hyaluronan (HA) oligosaccharides is reported. In particular, a titration experiment with increasing concentrations of octasaccharide (HA(8)) to protein produced a noncovalent complex with 1:1 stoichiometry when the oligosaccharide was in molar excess. However, in the presence of a molar excess of tetrasaccharide (HA(4)) nearly all proteins and oligosaccharides were observed in their unbound charge states. These results are consistent with solution-phase properties for this interaction in which HA(8), but not HA(4), supports high affinity Link_TSG6 binding. Hydrogen/deuterium amide exchange mass spectrometry (H/D-EX MS) was also utilized to investigate the level of global deuterium incorporation, over time, for Link_TSG6 in both the absence and presence of HA(8). After dilution into quenching conditions, deuterium incorporation reached limiting asymptotic values of 37 and 26 deuterons for the free and bound protein at 240 and 480 min, respectively, indicating that the oligosaccharide interferes with amide exchange on binding. To detect sequence-specific deuterium incorporation, pepsin digestion of Link_TSG6 in both the absence and presence of HA(8) was performed. A level of deuterium incorporation of 10-30% was observed for peptides analyzed in free Link_TSG6. Interestingly, HA(8) blocked some sites of proteolysis in Link_TSG6 compared to the free protein. Molecular modeling indicated that amino acids proximal to the ligand correlated with regions of the protein that were resistant to enzymatic digestion. Of the peptides that could be analyzed by H/D-EX MS in the presence of the ligand, a 30-60% reduction in deuterium incorporation, relative to the free protein, was observed, even for those sequences not directly involved in HA binding. These results support the utility of FTMS as a method for the characterization of protein-carbohydrate interactions.     

Matrix-assisted laser desorption ionization hydrogen/deuterium exchange studies to probe peptide conformational changes

Hydrogen/deuterium (H/D) exchange chemistry monitored by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is used to study solution phase conformational changes of bradykinin, alpha-melanocyte stimulating hormone, and melittin as water is added to methanol-d4, acetonitrile, and isopropanol-d8 solutions. The results are interpreted in terms of a preference for the peptides to acquire more compact conformations in organic solvents as compared to the random conformations. Our interpretation is supported by circular dichroism spectra of the peptides in the same solvent systems and by previously published structural data for the peptides. These results demonstrate the utility of MALDI-TOF as a method to monitor the H/D exchange chemistry of peptides and investigations of solution-phase conformations of biomolecules.     

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.     

Site-specific amide hydrogen/deuterium exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry

Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach. In this study, site-specific amide deuterium content was measured in oxidized and reduced E. coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D(2)O phosphate-buffered solution (pH 5.7). The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants. The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data. In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels. Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences. The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms. The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.     

Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.     

Higher order structure characterization of protein therapeutics by hydrogen/deuterium exchange mass spectrometry

Characterization of therapeutic drugs is a crucial step in drug development in the biopharmaceutical industry. Analysis of protein therapeutics is a challenging task because of the complexities associated with large molecular size and 3D structures. Recent advances in hydrogen/deuterium-exchange mass spectrometry (HDX-MS) have provided a means to assess higher-order structure of protein therapeutics in solution. In this review, the principles and procedures of HDX-MS for protein therapeutics characterization are presented, focusing on specific applications of epitope mapping for protein–protein interactions and higher-order structure comparison studies for conformational dynamics of protein therapeutics. Figure

HDX of protein backbone amide hydrogen     

Gas-phase hydrogen/deuterium exchange of adenine nucleotides

Gas-phase hydrogen/deuterium exchange reactions of (de)protonated (sodiated) adenosine-5'-mono-, di- and triphosphate ions with CD(3)OD, CD(3)CO(2)D and ND(3) were achieved using a combination of electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry. The reaction kinetics are dependent on factors such as the charge state, the phosphate chain length, the properties of the exchange reactants and the sodium content. The results indicate that the overall H/D exchange may involve specific sites even if endowed with high energetic barriers. The enhanced reactivity exhibited by adenosine polyphosphate ions compared with adenosine-5'-monophosphate suggests a critical role of the polyphosphate chain in rendering conformationally accessible remote H-donor sites. Low-energy collision-induced dissociation of (sodiated) adenine nucleotides anions supports the aptitude of the (poly)phosphate chain in probing distant sites via the intermediacy of a cyclic structure.     

Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification

Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH(2))-CO-N(R2)-CH(2)-S-R3) led to the identification of five metabolites (M1-M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H(2)O and D(2)O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated.     

Normal carbon acid referencing for protein amide hydrogen exchange

Measurement of the exchange kinetics for amide hydrogens along the protein backbone continues to offer valuable insights into structural stability and conformational dynamics. Since such studies routinely compare samples that differ in solution conditions or mechanical handling, normalization of the relative exchange rates can present a potentially significant source of experimental uncertainty. The carbon acids 1,3-dimethylimidazolium cation and thiomethylacetonitrile exhibit base catalyzed exchange rates similar to those of the slowly exchanging amides, under conditions typical for protein studies. With 13C enrichment at the acidic carbon position to facilitate selective observation, such carbon acids offer practical internal calibration of exchange.     

The determination of high-affinity protein/inhibitor binding constants by electrospray ionization hydrogen/deuterium exchange mass spectrometry

Recently, a hydrogen/deuterium exchange method termed SUPREX (Stability of Unpurified Proteins from Rates of hydrogen/deuterium EXchange), capable of measuring protein/ligand binding constants, which utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported. Unlike more conventional approaches, SUPREX is inherently capable of measuring Kd values of tight binding ligands. Here we present a SUPREX-based method, incorporating automation and electrospray ionization (ESI)-MS, to measure Kd values for very potent inhibitors of the kinase PKCtheta. The use of ESI offers an alternative to MALDI, with the advantages of improved mass measurement precision for larger proteins, and amenability to automation. Kd values generated by this method are in good agreement with those generated by a molecular protein kinase assay.     

Improving hydrogen/deuterium exchange mass spectrometry by reduction of the back-exchange effect

The measurement of deuterium incorporation kinetics using hydrogen/deuterium (H/D) exchange experiments is a valuable tool for the investigation of the conformational dynamics of biomolecules in solution. Experiments consist of two parts when using H/D exchange mass spectrometry to analyse the deuterium incorporation. After deuterium incorporation at high D(2)O concentration, it is necessary to decrease the D(2)O concentration before the mass analysis to avoid deuterium incorporation under artificial conditions of mass spectrometric preparation and measurement. A low D(2)O concentration, however, leads to back-exchange of incorporated deuterons during mass analysis. This back-exchange is one of the major problems in H/D exchange mass spectrometry and must be reduced as much as possible. In the past, techniques using electrospray ionization (ESI) had the lowest back-exchange values possible in H/D exchange mass spectrometry. Methods for the measurement of H/D exchange by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that have been developed since 1998 have some significant advantages, but they could not achieve the back-exchange minima of ESI methods. Here, we present a protocol for H/D exchange MALDI-MS which allows for greater minimization of back-exchange compared with H/D exchange ESI-MS under similar conditions.     

Automatic analysis of hydrogen/deuterium exchange mass spectra of peptides and proteins using calculations of isotopic distributions

High mass-resolving power has been shown to be useful for studying the conformational dynamics of proteins by hydrogen/deuterium (H/D) exchange. A computer algorithm was developed that automatically identifies peptides and their extent of deuterium incorporation from H/D exchange mass spectra of enzymatic digests or fragment ions produced by collisionally induced dissociation (CID) or electron capture dissociation (ECD). The computer algorithm compares measured and calculated isotopic distributions and uses a fast calculation of isotopic distributions using the fast Fourier transform (FFT). The algorithm facilitates rapid and automated analysis of H/D exchange mass spectra suitable for high-throughput approaches to the study of peptide and protein structures. The algorithm also makes the identification independent on comparisons with undeuterated control samples. The applicability of the algorithm was demonstrated on simulated isotopic distributions as well as on experimental data, such as Fourier transform ion cyclotron resonance (FTICR) mass spectra of myoglobin peptic digests, and CID and ECD spectra of substance P.     

Conformational changes in Akt1 activation probed by amide hydrogen/deuterium exchange and nano‐electrospray ionization mass spectrometry

Amide hydrogen exchange coupled to nano‐electrospray ionization mass spectrometry (nano‐ESI‐MS) has been used to identify and characterize localized conformational changes of Akt upon activation. Active or inactive Akt was incubated in D₂O buffer, digested with pepsin, and analyzed by nano‐ESI‐MS to determine the deuterium incorporation. The hydrogen/deuterium (H/D) exchange profiles revealed that Akt undergoes considerable conformational changes in the core structures of all three individual domains after activation. In the PH domain, four β‐strand (β1, β2 β5 and β6) regions containing membrane‐binding residues displayed higher solvent accessibility in the inactive state, suggesting that the PH domain is readily available for the binding to the plasma membrane for activation. In contrast, these β‐strands became less exposed or more folded in the active form, which is favored for the dissociation of Akt from the membrane. The beginning α‐helix J region and the C‐terminal locus (T450‐470P) of the regulatory domain showed less folded structures that probably enable substrate entry. Our data also revealed detailed conformational changes of Akt in the kinase domain due to activation, some of which may be attributed to the interaction of the basic residues with phosphorylation sites. Our H/D exchange results indicating the conformational status of Akt at different activation states provided new insight for the regulation of this critical protein involved in cell survival. Published in 2009 by John Wiley & Sons, Ltd.     

Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry—A review

Assessing the functional outcome of protein interactions in structural terms is a goal of structural biology, however most techniques have a limited capacity for making structure–function determinations with both high resolution and high throughput. Mass spectrometry can be applied as a reader of protein chemistries in order to fill this void, and enable methodologies whereby protein structure–function determinations may be made on a proteome-wide level. Protein hydrogen/deuterium exchange (H/DX) offers a chemical labeling strategy suitable for tracking changes in “dynamic topography” and thus represents a powerful means of monitoring protein structure–function relationships. This review presents the exchange method in the context of interaction analysis. Applications involving interface detection, quantitation of binding, and conformational responses to ligation are discussed, and commentary on recent analytical developments is provided.     

Denaturant sensitive regions in creatine kinase identified by hydrogen/deuterium exchange

The GdmHCl-induced unfolding of creatine kinase (CK) has been studied by hydrogen/deuterium (H/D) exchange combined with mass spectrometry. MM-CK unfolded for various periods in different denaturant concentrations was pulsed-labeled with deuterium to identify different conformational intermediate states. For all denaturation times or GdmHCl concentrations, we observed variable proportions of only two species. The low-mass envelope of isotope peaks corresponds to a species that has gained about 10 deuteriums more than native CK, and the high-mass envelope to a completely deuterated species. To localize precisely the unfolded regions in the states highly populated during denaturation, the protein was digested with two proteases (pepsin and type XIII protease) after H/D exchange and rapid quenching of the reaction. The two sets of fragments obtained were analyzed by liquid chromatography coupled to mass spectrometry to determine the deuterium level in each fragment. Bimodal distributions of deuterium were found for most peptides, indicating that these regions were either folded or unfolded. This behavior is consistent with cooperative, localized unfolding. However, we observed a monomodal distribution of deuterium in two regions (1-12 and 162-186). We conclude that the increment of mass observed in the low-mass species of the intact protein (+10 Da) has its origin in these two segments. These regions, which are very sensitive to low GdmHCl concentrations, are involved in the monomer-monomer interface of CK and their perturbation is likely to weaken the dimeric structure. At higher denaturant concentration, this would induce dissociation of the dimer.     

Gas phase hydrogen/deuterium exchange reactions of fluorophenyl anions

Hydrogen/deuterium (H/D) exchange reactions of fluorophenyl and difluorophenyl anions (C₆H₄F^?, o-C₆H₃F ₂ ^? , m-C₆H₃F ₂ ^? , p-C₆H₃F ₂ ^? ) have been studied using the flowing afterglow-selected ion flow tube technique. The C₆H₄F^? anion exchanges all hydrogens for deuterium upon reaction with D₂O. The difluorophenyl anions o-, m-, and p-C₆H₃F ₂ ^? exchange three, two, and one hydrogen, respectively, with D₂O, whereas they undergo one, two, and three H/D exchanges, respectively, with CH₃OD. The structures of the anions and the isotope exchange dynamics within the intermediate ion-dipole complexes are discussed using ab initio molecular orbital calculations. Calculated values for the proton affinities of the most stable anions are 385.2, 378.0, 371.9, and 378.2 kcal/mol for C₆H₄F^?, o-C₆H₃F ₂ ^? , m-C₆H₃F ₂ ^? , and p-C₆H₃F ₂ ^? , respectively, in excellent agreement (within 2 kcal/mol) with the previous experimental values for the acidities of the corresponding fluorobenzenes. The H/D exchange results are explained by the energy differences of the intermediate DO^? and CH₃O^? species within the ion-dipole complexes; CH₃O^? is mobile within the “hot” intermediate complex, whereas DO^? is nearly “frozen” within the complex and cannot migrate across the barriers caused by the fluorine atoms or by the π electrons.     

Rate and extent of gas-phase hydrogen/deuterium exchange of bradykinins: evidence for peptide zwitterions in the gas phase

The gas-phase H/D exchange of bradykinin [M + H]⁺, [M + Na]⁺, [M + 2H]²⁺, and [M + H + Na]²⁺ ions; des-Arg¹-bradykinin, des-Arg⁹-bradykinin, and bradykinin fragment 2-7 [M + H]⁺ ions; and O-methylbradykinin [M + H]⁺ and [M + 2H]²⁺ ions with D₂O have been examined by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at 9.4 T. The different peptides vary widely in exchange rate and extent of deuterium incorporation. H/D exchange was slowest and deuterium incorporation was least for bradykinin [M + H]⁺, [M + H + Na]²⁺ and bradykinin methyl ester [M + 2H]²⁺ ions. In contrast, H/D exchange and extent of deuteration are higher for des-Arg¹-bradykinin, des-Arg⁹-bradykinin, and bradykinin fragment 2-7 [M + H]⁺ ions; and highest for bradykinin [M + Na]⁺ and [M + 2H]²⁺, and O-methylbradykinin [M + H]⁺. Because the most likely site of protonation is the guanidino group of arginine, the above reactivity pattern strongly supports a zwitterion form for protonated gas-phase bradykinin.