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.     

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.     

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.     

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.     

Relaxation compensation in chemical exchange measurements for the quantitation of amide hydrogen exchange in larger proteins

The increasingly rapid transverse relaxation of larger macromolecules serves to limit the practical length of various types of mixing periods. When chemical exchange dynamics are used to determine the rates of amide hydrogen exchange with the bulk solvent, the foreshortened mixing period results in lowered sensitivity. Three approaches are examined for increasing the practical length of the mixing period. The transverse relaxation rate of the amide resonances is decreased by perdeuteration of the carbon‐bound hydrogen positions and also by introduction of a TROSY‐based ¹⁵N‐separated pulse sequence. Reference experiments are proposed which provide accurate compensation for relaxation effects so that exchange rate data can be obtained over the entire mixing period profile. As a result, more than a 100‐fold range of amide exchange rates can be accurately determined for a moderate‐sized protein. Copyright © 2003 John Wiley & Sons, Ltd.     

Dependence of amide vibrations on hydrogen bonding

The effect of hydrogen bonding on the amide group vibrational spectra has traditionally been rationalized by invoking a resonance model where hydrogen bonding impacts the amide functional group by stabilizing its [(-)O-C=NH (+)] structure over the [O=C-NH] structure. However, Triggs and Valentini's UV-Raman study of solvation and hydrogen bonding effects on epsilon-caprolactum, N, N-dimethylacetamide (DMA), and N-methylacetamide (NMA) ( Triggs, N. E.; Valentini, J. J. J. Phys. Chem. 1992, 96, 6922-6931) casts doubt on the validity of this model by demonstrating that, contrary to the resonance model prediction, carbonyl hydrogen bonding does not impact the AmII' frequency of DMA. In this study, we utilize density functional theory (DFT) calculations to examine the impact of hydrogen bonding on the C=O and N-H functional groups of NMA, which is typically used as a simple model of the peptide bond. Our calculations indicate that, as expected, the hydrogen bonding frequency dependence of the AmI vibration predominantly derives from the C=O group, whereas the hydrogen bonding frequency dependence of the AmII vibration primarily derives from N-H hydrogen bonding. In contrast, the hydrogen bonding dependence of the conformation-sensitive AmIII band derives equally from both C=O and N-H groups and thus, is equally responsive to hydrogen bonding at the C=O or N-H site. Our work shows that a clear understanding of the normal mode composition of the amide vibrations is crucial for an accurate interpretation of the hydrogen bonding dependence of amide vibrational frequencies.     

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.     

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.     

Fragmentation of mycosporine-like amino acids by hydrogen/deuterium exchange and electrospray ionisation tandem mass spectrometry

The determination and identification of mycosporine-like amino acids (MAAs) from algae remain a major challenge due to the low concentration. Mass spectrometry (MS) can make an invaluable contribution in the search and identification of MAAs because of its high sensitivity, possibility of coupling with liquid chromatography, and the availability of powerful tandem mass spectrometric techniques. However, the unequivocal determination of the presence and location of important functional groups present on the basic skeleton of the MAAs is often elusive due to their inherent instability under MS conditions. In this study, the use of hydrogen/deuterium (H/D) exchange and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) for characterisation of four MAAs (palythine, asterina, palythinol and shinorine) isolated from the macroalgae Gracilaria tenuistipitata Chang et Xia was investigated. The accurate-mass confirmation of the protonated molecules was performed on a Q-TOF instrument. We demonstrate that employing deuterium labelling in ESI-MS/MS analysis provides a convenient tool for the determination of new MAAs. Although the fragmentation patterns of MAAs were discussed earlier, to our knowledge, this is the first time that mechanisms are proposed.     

Comparison of continuous and pulsed labeling amide hydrogen exchange/mass spectrometry for studies of protein dynamics

In contrast to the rigid structures portrayed by X-ray diffraction, proteins in solution display constant motion which leads to populations that are momentarily unfolded. To begin to understand protein dynamics, we must have experimental methods for determining rates of folding and unfolding, as well as for identifying structures of folding and unfolding intermediates. Amide hydrogen exchange has become an important tool for such measurements. When urea is used to stabilize unfolded forms of proteins, the refolding rates may become slower than the rates of isotope exchange. In such cases, the intermolecular distribution of deuterium among the entire population of molecules may become bimodal, giving rise to a bimodal distribution of isotope peaks in mass spectra of the protein or its peptic fragments. When the protein is exposed continuously to D2O, the relative intensities of the two envelopes of isotope peaks give an integrated account of populations participating in the folding/unfolding process. However, when the protein is exposed only briefly to D2O, the relative intensities of the two envelopes of isotope peaks give an instantaneous measure of the folded/unfolded populations. Application of these two labeling methods to a large protein, aldolase, is described along with a discussion of specific parameters required to optimize these experiments.     

Protein adsorption-desorption on electrospray capillary walls--no influence on aggregate distribution

Adsorbed proteins on walls of glass capillaries used for electrospray (ES) can desorb and potentially affect size distributions and, thus, quantification of aggregates of proteins. In this study we use differential mobility analysis (DMA) to investigate the size distribution of various proteins eluting from bare and passivated glass capillaries. We found no significant differences in aggregate distributions from unpassivated capillaries at 'steady state' when compared to aggregate distributions from passivated capillaries implying that desorbing proteins do not influence protein aggregate distribution. Surface passivation with gelatin was found to be considerably more effective in limiting adsorption of two antibodies (Rituxan and polyclonal human IgG) compared to passivation with BSA. Gelatin passivation was also found to be stable for a few days and from a pH range of 4.8-9.0.     

On amide exchange reaction of polymide precursor

The reaction of poly(amic acid) (PAA) derived from pyromellitic dianhydride (PMDA) and oxydianiline (ODA) with 1-aminopyrene (APy) in solution as a model of amide exchange reaction between PAAs was studied in the temperature range of 0–60°C using viscometry and light-scattering (LS) measurements. The decrease in the weight-average molecular weight (M?_w) of PAA in N,N-dimethylacetamide (DMAc) solution with time and the acceleration of M?_w drop due to the increase in storage temperature or the addition of APy into PAA solution were observed. Apparent activation energies (E_a) for scission of PAA chains were similar: about 13 kcal/mol in PAA/DMAc and PAA/APy/DMAc, respectively. When stored at 60°C for a week, the number of scissions per polymer chain in PAA/DMAc is about 2, but is about 5 in PAA/DMAc with a large amount of APy. The result indicates that the M?_w drop accelerated by the addition of APy is attributed to amide exchange reaction between PAA Chains and monofunctional APy. It was concluded from the dependence of M?_w drop on Apy concentration that the exchange reaction between different PAA molecules during storage of PAA/PAA solution may scarcely occur under the conditions (storage time and temperature) used for preparation of PAA/PAA blends.     

Effects of internal hydrogen bonds between amide groups: protonation of alicyclic diamides

The proton affinity (PA) of cyclopentane carboxamide 1, cyclohexane carboxamide 2 and their secondary and tertiary amide derivatives S1, S2, T1 and T2, was determined by the thermokinetic method and the kinetic method [PA(1) = 888 +/- 5 kJ mol(1); PA(2) = 892 +/- 5 kJ mol(1); PA(S1) = 920 +/- 6 kJ mol(1); PA(S2) = 920 +/- 6 kJ mol(1); PA(T1) = 938 +/- 6 kJ mol(1); PA(T2) = 938 +/- 6 kJ mol(1)]. Special entropy effects are not observed. Additionally, the effects of protonation have been studied using an advanced kinetic method for all isomers 37 of cyclopentane dicarboxamides and cyclohexane dicarboxamides (with the exception of cis-cyclopentane-1,2-dicarboxamide) and their bis-tertiary derivatives T3T7 by estimating the PA and the apparent entropy of protonation Delta(DeltaS(app)). Finally, the study was extended to bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxamide 8 and its bis-tertiary derivative T8, to all stereoisomers of bicyclo[2.2.1]heptane-2,3-dicarboxamide 9, their secondary and tertiary amide derivatives S9 and T9, and to endoendobicyclo[2.2.1]heptane-2,5-dicarboxamide 10 and the corresponding secondary and tertiary derivatives S10 and T10. Compared with 1 and 2, all alicyclic diamides exhibit a significant increase of the PA (DeltaPA) and special entropy effects on protonation. For alicyclic diamides, which can not accommodate a conformation appropriate for building a proton bridge, the values of DeltaPA and Delta(DeltaS(app)) are small to moderate. This is explained by ion / dipole interactions between the protonated and neutral amide group which stabilize the protonated species but hinder the free rotation of the amide groups. If any of the conformations of the alicyclic diamide allows formation of a proton bridge, DeltaPA and Delta(DeltaS(app)) increase considerably. A spectacular case is cis-cyclohexane-1,4-dicarboxamide 7c which is the most basic monocyclic diamide, although generation of the proton bridge requires the unfavorable boat conformation with both amide substituents at a flagpole position. A pre-orientation of the two amide groups in such a 1,4-position in 10 results in a particularly large PA of < 1000 kJ mol(1). The observation of comparable values for Delta(DeltaS(app)) for linear and monocyclic diamides indicates that a major part of the entropy effects originates from freezing the free rotation of the amide groups by formation of the proton bridge. This is corroborated by observing corresponding effects during the protonation of dicarboxamides containing the rigid bicyclo[2.2.1]heptane carbon skeleton, where the only internal movements of the molecules corresponds to rotation of the amide substituents.     

In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D2O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H2O or D2O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site(s) of modification for each metabolite.     

Observation of amide anions in solution by electrospray ionization mass spectrometry

Negative quasimolecular ions of aromatic carboxylic acid amides have been observed unexpectedly under electrospray ionization conditions. Hypothetically, deprotonation of either carboxamide or carboximidic acid tautomers can produce anions with equivalent resonance structures, the stability of which is affected by conjugated aromatic substituents. In this study, a series of meta and para substituted benzamides were analyzed using electrospray ionization mass spectrometry in aqueous methanolic solutions. The degree of ionization was found to be pH dependent and was enhanced by electron-withdrawing substituents and suppressed by electron-donating groups. The observed effect on apparent acidity can be accounted for by resonance stabilization.     

Inter- and intra-molecular migration of peptide amide hydrogens during electrospray ionization

The isotopic exchange of amide hydrogens in proteins in solution strongly depends on the surrounding protein structure, thereby allowing structural studies of proteins by mass spectrometry. However, during electrospray ionization (ESI), gas phase processes may scramble or deplete the isotopic information. These processes have been investigated by on-line monitoring of the exchange of labile deuterium atoms in homopeptides with hydrogens from a solvent suitable for ESI. The relative contribution of intra- and inter-molecular exchange in the gas phase could be studied from their distinct influence on the well-characterized exchange processes in the spraying solution. The deuterium content of individual labile hydrogens was assessed from the isotopic patterns of two consecutive collision-induced dissociation fragments, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Results demonstrate that gas phase exchange in the high-pressure region between the capillary and the skimmer cause substantial depletion of the isotopic information of penta-phenylalanine and penta-aspartic acid. For penta-alanine and hexa-tyrosine, the amide hydrogens located close to the N-terminus are depleted from deuterium during mass analysis. Amide hydrogens located close to the C-terminus still retain the information of the isotopic state in solution, but they are redistributed by intra-molecular exchange of the amide hydrogens with the C-terminal hydroxyl group.     

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.