Protein–protein interaction dynamics by amide H/H exchange mass spectrometry

Amide H/²H exchange detected by mass spectrometry provides a powerful tool for observing changes that occur upon protein–protein interactions. In general, it is possible to observe protection of surface amides, they become less solvent exposed when they are buried at the interface. The information thus obtained about the location of the protein–protein interface is useful for building a correct docked structure of the protein–protein complex. Examples of protein–protein interfaces that were correctly identified by such methods include the thrombin–thrombomodulin interaction and the interaction between the regulatory and catalytic subunit of protein kinase A. Amide exchange also affords a view into the subtle changes in the ensemble of states that occur upon protein modification or protein–protein binding. Examples of proteins in which amide exchange has been used to observe phosphorylation-induced changes include ERK2 and CheB. Amide exchange showed the pathway of communication between the cAMP-binding site and the catalytic subunit site within the regulatory subunit of protein kinase A. Clues as to how thrombomodulin regulates the catalytic activity of thrombin were also obtained.     

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.     

Optimization of conditions for studies of protein unfolding by hydrogen exchange/mass spectrometry

Understanding the forces driving protein folding and aggregation is an essential step in developing means for controlling these important processes. Amide hydrogen exchange, coupled with mass spectrometry, has become an important method for studying protein unfolding and refolding. To extend procedures developed to study unfolding of relatively soluble proteins to less soluble, aggregation-prone proteins requires special considerations. This publication describes a general strategy developed using yeast transaldolase, which aggregates easily under conditions required to study its unfolding. Results presented here show that reducing the protein concentration to the nanomolar range is essential for managing aggregation of transaldolase. In addition, the present results point to use of relatively high concentrations of denaturants and short incubation times to minimize aggregation. These results also show how amide hydrogen exchange, coupled with mass spectrometry, can be used to study soluble aggregates.     

Hydrogen exchange mass spectrometry for studying protein structure and dynamics

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in solution. This approach relies on the fact that exposure of a protein to D(2)O induces rapid amide H → D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a number of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissociation methods provide a solution to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful reference for practitioners, who require an overview of some recent trends in HDX/MS.     

Analysis of protein complexes with hydrogen exchange and mass spectrometry

Analysis of protein complexes using hydrogen exchange (HX) combined with high resolution electrospray mass spectrometry (MS) is demonstrated. HX MS offers the possibility to analyze the strength of binding in protein complexes, to identify regions that undergo binding induced structural changes, and to study the nature (hydrophobic, electrostatic, etc.) of binding between two or more proteins. In the current work, a heteromeric complex containing UBC9 (an E2 conjugating enzyme) and SUMO-1 (a ubiquitin-like modifier) was investigated by incubating the complex in D2O and measuring the amount of deuterium incorporation with MS. SUMO-1 had significant changes in deuterium levels when bound to UBC9. In contract, few or no changes in deuterium levels were detected in UBC9 when part of the complex, even at the binding interface. Titrations were used to estimate the binding constant for the complex. The nature of the interface was probed by creating a site-directed mutant form of UBC9. The mutant form showed no detectable binding to SUMO-1 and thereby suggested that binding between these two proteins is primarily electrostatically driven. This application of HX MS demonstrates its value in the study of protein complexes and protein machinery.     

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.     

Probing protein dynamics and function under native and mildly denaturing conditions with hydrogen exchange and mass spectrometry

A combination of hydrogen exchange and mass spectrometry emerged in recent years as a powerful experimental tool capable of probing both structural and dynamic features of proteins. Although its concept is very simple, the interpretation of experimental data is not always straightforward, as a combination of chemical reactions (isotope exchange) and dynamic processes within protein molecules give rise to convoluted exchange patterns. This paper provides a historical background of this technique, candid assessment of its current state and limitations and a discussion of promising recent developments that can result in tremendous improvements and a dramatic expansion of the scope of its applications.     

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.     

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     

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.     

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.     

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.     

HDXFinder: automated analysis and data reporting of deuterium/hydrogen exchange mass spectrometry

Hydrogen/deuterium exchange in combination with mass spectrometry (H/D MS) is a sensitive technique for detection of changes in protein conformation and dynamics. However, wide application of H/D MS has been hindered, in part, by the lack of computational tools necessary for efficient analysis of the large data sets associated with this technique. We report a novel web-based application for automatic analysis of H/D MS experimental data. This application relies on the high resolution of mass spectrometers to extract all isotopic envelopes before correlating these envelopes with individual peptides. Although a fully automatic analysis is possible, a variety of graphical tools are included to aid in the verification of correlations and rankings of the isotopic peptide envelopes. As a demonstration, the rate constants for H/D exchange of peptides from rabbit muscle pyruvate kinase are mapped onto the structure of this protein.     

Inverse 15N-metabolic labeling/mass spectrometry for comparative proteomics and rapid identification of protein markers/targets

The inverse labeling/mass spectrometry strategy has been applied to protein metabolic (15)N labeling for gel-free proteomics to achieve the rapid identification of protein markers/targets. Inverse labeling involves culturing both the perturbed (by disease or by a drug treatment) and control samples each in two separate pools of normal and (15)N-enriched culture media such that four pools are produced as opposed to two in a conventional labeling approach. The inverse labeling is then achieved by combining the normal (14)N-control with the (15)N-perturbed sample, and the (15)N-control with the (14)N-perturbed sample. Both mixtures are then proteolyzed and analyzed by mass spectrometry (coupled with on-line or off-line separation). Inverse labeling overcomes difficulties associated with protein metabolic labeling with regard to isotopic peak correlation and data interpretation in the single-experiment approach (due to the non-predictable/variable mass difference). When two data sets from inverse labeling are compared, proteins of differential expression are readily recognized by a characteristic inverse labeling pattern or apparent qualitative mass shifts between the two inverse labeling analyses. MS/MS fragmentation data provide further confirmation and are subsequently used to search protein databases for protein identification. The methodology has been applied successfully to two model systems in this study. Utilizing the inverse labeling strategy, one can use any mass spectrometer of standard unit resolution, and acquire only the minimum, essential data to achieve the rapid and unambiguous identification of differentially expressed protein markers/targets. The strategy permits quick focus on the signals of differentially expressed proteins. It eliminates the detection ambiguities caused by the dynamic range of detection. Finally, inverse labeling enables the detection of covalent changes of proteins responding to a perturbation that one might fail to distinguish with a conventional labeling experiment.     

Electrospray ionization mass spectrometry and hydrogen/deuterium exchange for probing the interaction of calmodulin with calcium

The extent of H/D exchange of the protein calmodulin in solution was monitored by mass spectrometry following electrospray ionization (ESI) of the protein. In the absence of Ca2+, approximately 115 protons are exchanged for deuteriums after 60 min. As the calmodulin is titrated with Ca2+, the extent of exchange decreases significantly (i.e., by 24 protons), indicating Ca(2+)-induced folding of the protein to a tighter, less solvent-accessible form. The extent of H/D exchange ceases to decrease when the amount of added Ca2+ is sufficient to convert greater than 80% of the calmodulin to a form bound by four calcium ions. Lysozyme, a protein of similar molecular weight, does not show a significant decrease in the extent of H/D exchange as it binds to Ca2+, indicating that the changes in H/D exchange for calmodulin reflect tertiary structural change that occur upon binding with Ca2+.     

In-situ monitoring of protein labeling reactions by matrix-assisted laser desorption/ionization mass spectrometry

Taking the labeling reaction of horse heart cytochrome c or ubiquitin with biotinamidocaproate N-hydroxysucchinimide ester (biotin-NHS) as test cases, this report demonstrates the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for in-situ monitoring of the labeling process and for determining the composition of the labeled products without the need for prior separation. The effects of pH and starting materials concentration on the labeling process were investigated in detail. Our MALDI MS results show that: (1) labeled products are always mixtures of different conjugates, which may explain peak broadening found in chromatographic studies of labeling reactions; (2) the higher conjugate fractions become more prominent as the labeling reaction proceeds, with a concomitant exponential decline of the lower conjugate fractions; (3) biotin-NHS can be incorporated into peptides and protein in a stepwise and controlled manner simply by adjusting the molar ratio of the starting materials.     

In-situ monitoring of protein labeling reactions by matrix-assisted laser desorption/ionization mass spectrometry

Taking the labeling reaction of horse heart cytochrome c or ubiquitin with biotinamidocaproate N-hydroxysucchinimide ester (biotin-NHS) as test cases, this report demonstrates the usefulness of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for in-situ monitoring of the labeling process and for determining the composition of the labeled products without the need for prior separation. The effects of pH and starting materials concentration on the labeling process were investigated in detail. Our MALDI MS results show that: (1) labeled products are always mixtures of different conjugates, which may explain peak broadening found in chromatographic studies of labeling reactions; (2) the higher conjugate fractions become more prominent as the labeling reaction proceeds, with a concomitant exponential decline of the lower conjugate fractions; (3) biotin-NHS can be incorporated into peptides and protein in a stepwise and controlled manner simply by adjusting the molar ratio of the starting materials.     

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.     

Characterization of intermolecular beta-sheet peptides by mass spectrometry and hydrogen isotope exchange

The self-assembly of beta-sheet peptide domains resulting in the formation of fibrillar aggregates (amyloids) is a feature of various neurodegenerative disorders. In order to evaluate mass spectrometric methods for the characterization of intermolecular beta-sheet structures the hydrogen/deuterium exchange behaviour of model peptides DPKGDPKG-(VT)(n)-GKGDPKPD-amide (n = 3,4,5,6,7,8), (VT)(n)-peptides, composed of a central beta-sheet-forming domain and N- and C-terminal nonstructured octapeptide sequences, was measured by electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The kinetic analysis of the hydrogen/deuterium exchange (HX) shows that intermolecular beta-sheet structures contain slowly exchanging protons (k 相似文献