Hydrogen deuterium exchange mass spectrometry (HDX‐MS) is a powerful technique for studying protein dynamics, which is an important factor governing protein functions. However, the process of hydrogen/deuterium exchange (HDX) of proteins is highly complex and the underlying mechanism has not yet been fully elucidated. Meanwhile, molecular dynamics (MD) simulation is a computational technique that can be used to elucidate HDX behaviour on proteins and facilitate interpretation of HDX‐MS data. This article aims to summarize the current understandings on the mechanism of HDX and its correlation with MD simulation, to discuss the recent developments in the techniques of HDX‐MS and MD simulation and to extend the perspectives of these two techniques in protein dynamics study. 相似文献
Mass spectrometry (MS) plays a central role in studies on protein structure and dynamics. This review highlights some of the recent developments in this area, with focus on applications involving the use of electrospray ionization (ESI) MS. Although this technique involves the transformation of analytes into highly nonphysiological species (desolvated gas-phase ions in the vacuum), ESI-MS can provide detailed insights into the solution-phase behavior of proteins. Notably, the ionization process itself occurs in a structurally sensitive manner. An increased degree of solution-phase unfolding is correlated with a higher level of protonation. Also, ESI allows the transfer of intact noncovalent complexes into the gas phase, thereby yielding information on binding partners, stoichiometries, and even affinities. A particular focus of this article is the use of hydrogen/deuterium exchange (HDX) methods and hydroxyl radical (.OH) labeling for monitoring dynamic and structural aspect of solution-phase proteins. Conceptual similarities and differences between the two methods are discussed. We describe a simple method for the computational simulation of protein HDX patterns, a tool that can be helpful for the interpretation of isotope exchange data recorded under mixed EX1/EX2 conditions. Important aspects of .OH labeling include a striking dependence on protein concentration, and the tendency of commonly used solvent additives to act as highly effective radical scavengers. If not properly controlled, both of these factors may lead to experimental artifacts. 相似文献
The initial stages of drug discovery are increasingly reliant on development and improvement of analytical methods to investigate protein-protein and protein-ligand interactions. For over 20 years, mass spectrometry (MS) has been recognized as providing a fast, sensitive and high-throughput methodology for analysis of weak non-covalent complexes. Careful control of electrospray ionization conditions has enabled investigation of the structure, stability and interactions of proteins and peptides in a solvent free environment. This critical review covers the use of mass spectrometry for kinetic, dynamic and structural studies of proteins and protein complexes. We discuss how conjunction of mass spectrometry with related techniques and methodologies such as ion mobility, hydrogen-deuterium exchange (HDX), protein footprinting or chemical cross-linking can provide us with structural information useful for drug development. Along with other biophysical techniques, such as NMR or X-ray crystallography, mass spectrometry provides a powerful toolbox for investigation of biological problems of medical relevance (204 references). 相似文献
PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating
half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we
applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze granulocyte colony stimulating factor (G-CSF) upon
PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible
and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF.
The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although
the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The
data complexity often encountered in HDX MS measurements was greatly reduced through a data processing and presentation format
designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies,
process monitoring, and protein therapeutic characterization in the biopharmaceutical industry. 相似文献
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase
deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements
are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide
resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished
by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique
electron transfer dissociation (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing
single-amide deuterium incorporation for differential HDX analysis of a nuclear receptor upon binding small molecule ligands.
We are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established
and fully automated HDX platform making differential single-amide HDX possible for bottom-up analysis of complex systems.
We have applied this system to determine differential single amide resolution HDX data for the peroxizome proliferator activated
receptor bound with two ligands of interest. 相似文献
Hydrogen/deuterium exchange (HDX) coupled to protein fragmentation either in solution (by means of proteolysis) or in the gas phase (using collisional activation of protein ions) and followed by mass spectral measurements of deuterium content of individual fragments has become one of the major experimental tools to probe protein structure and dynamics. One difficulty, which often arises in the course of interpretation of HDX MS data, is a need to separate deuterium contribution to the observed isotopic patterns from that of naturally occurring isotopes. Another frequently encountered problem, especially when HDX in solution is followed by protein ion fragmentation in the gas phase, is a need to determine the deuterium content of an internal protein segment based on the measured isotopic distributions of overlapping fragments. While several algorithms were developed in the past several years to address the first problem, the second one did not enjoy as much attention. Here we report a new algorithm based on a maximum entropy principle, which is capable of extracting local exchange data form the isotope distribution of overlapping fragments, as well as subtracting the background due to the presence of natural isotopes and residual deuterium in exchange buffers. The new method is tested with several proteins and appears to generate stable solutions even under unfavorable circumstances, e.g., when the resolving power of a mass analyzer is not sufficient to avoid signal interference or when the isotopic distributions of individual fragments are complex and cannot be approximated with simple binomial distributions. The latter feature makes the algorithm particularly useful when the exchange in solution is correlated or semicorrelated, paving the way to precise structural characterization of non-native protein states in solution. 相似文献
Determining the structure and dynamics of large biologically relevant molecules is one of the key challenges facing biology. Although X-ray crystallography (XRD) and nuclear magnetic resonance (NMR) yield accurate structural information, they are of limited use when sample quantities are low. Mass spectrometry (MS) on the other hand has been very successful in analyzing biological molecules down to atto-mole quantities and has hence begun to challenge XRD and NMR as the key technology in the life sciences. This trend has been further assisted by the development of MS techniques that yield structural information on biomolecules. Of these techniques, collision-induced dissociation (CID) and hydrogen/deuterium exchange (HDX) are among the most popular. Despite advances in applying these techniques, little direct experimental evidence had been available until recently to verify their proposed underlying reaction mechanisms. The possibility to record infrared spectra of mass-selected molecular ions has opened up a novel avenue in the structural characterization of ions and their reaction products. On account of its high pulse energies and wide wavelength tunability, the free electron laser for infrared experiments (FELIX) at FOM Rijnhuizen has been shown to be ideally suited to study trapped molecular ions with infrared photo-dissociation spectroscopy. In this paper, we review recent experiments in our laboratory on the infrared spectroscopic characterization of reaction products from CID and HDX, thereby corroborating some of the reaction mechanisms that have been proposed. In particular, it is shown that CID gives rise to linear fragment ion structures which have been proposed for some time, but also yields fully cyclical ring structures. These latter structures present a possible challenge for using tandem MS in the sequencing of peptides/proteins, as they can lead to a scrambling of the amino acid sequence information. In gas-phase HDX of an amino acid it is shown that the structure can be changed from a charge solvated to a zwitterionic structure, thereby demonstrating that HDX can be an invasive technique, in fact changing the structure of the analyte. These results emphasize that more fundamental work is required in order to understand the underlying mechanisms in two of the most important structural techniques in MS. 相似文献
In this report, a method for in-source hydrogen/deuterium (H/D) exchange at atmospheric pressure is reported. The method was named atmospheric pressure photo ionization hydrogen/deuterium exchange mass spectrometry (APPI HDX MS). H/D exchange was performed by mixing samples dissolved in toluene with CH3OD solvent and analyzing the mixture using atmospheric pressure photo ionization mass spectrometry (APPI-MS). The APPI HDX spectra obtained with contact times between the analyte solution and methanol-OD (CH3OD) of?<?0.5 s or 1 h showed the same pattern of H/D exchange. Therefore, it was concluded that APPI HDX occurred in the source but not in the solution. The proposed method does not require a specific type of mass spectrometer and can be performed at atmospheric pressure. H/D exchange can be performed in any laboratory with a mass spectrometer and a commercial APPI source. Using this method, multiple H/D exchanges of aromatic hydrogen and/or H/D exchange of active hydrogen were observed. These results demonstrated that H/D exchange can be used to distinguish between isomers containing primary, secondary, and tertiary amines, as well as pyridine and pyrrole functional groups.
The metastability of the native fold makes serpin (serine protease inhibitor) proteins prone to pathological conformational change, often by insertion of an extra β‐strand into the central β‐sheet A. How this insertion is made possible is a hitherto unresolved question. By the use of advanced hydrogen/deuterium‐exchange mass spectrometry (HDX‐MS) it is shown that the serpin plasminogen activator inhibitor 1 (PAI‐1) transiently unfolds under native condition, on a second‐to‐minute time scale. The unfolding regions comprise β‐strand 5A as well as the underlying hydrophobic core, including β‐strand 6B and parts of helices A, B, and C. Based thereon, a mechanism is proposed by which PAI‐1 makes transitions through progressively more unfolded states along the reaction coordinate to the inactive, so‐called latent form. Our results highlight the profound utility of HDX‐MS in detecting sparsely populated, transiently unfolded protein states. 相似文献
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. 相似文献
The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene–maleic acid lipid particle (SMALP) technology can be coupled with hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics. 相似文献
Hydrogen deuterium exchange (HDX) coupled to mass spectrometry (MS) is a well-established technique employed in the field of structural MS to probe the solvent accessibility, dynamics and hydrogen bonding of backbone amides in proteins. By contrast, fast photochemical oxidation of proteins (FPOP) uses hydroxyl radicals, liberated from the photolysis of hydrogen peroxide, to covalently label solvent accessible amino acid side chains on the microsecond-millisecond timescale. Here, we use these two techniques to study the structural and dynamical differences between the protein β2-microglobulin (β2m) and its amyloidogenic truncation variant, ΔN6. We show that HDX and FPOP highlight structural/dynamical differences in regions of the proteins, localised to the region surrounding the N-terminal truncation. Further, we demonstrate that, with carefully optimised LC-MS conditions, FPOP data can probe solvent accessibility at the sub-amino acid level, and that these data can be interpreted meaningfully to gain more detailed understanding of the local environment and orientation of the side chains in protein structures.
Trifluoroethanol (TFE)-induced conformational changes in dynorphin A (1-13) were investigated using charge-state distribution (CSD) and hydrogen-deuterium exchange (HDX), combined with electrospray ionization (ESI) mass spectrometry (MS). Individual amino acids involved in secondary structural elements were identified by collision-induced dissociation-tandem mass spectrometry (MS/MS). It was observed that dynorphin A (1-13) largely exists in an unfolded conformation and a folded structure in increasing concentrations of TFE. In 50% TFE, it forms an alpha-helix that encompasses residues 1-9 and remains flexible from residues 10 to 13. 相似文献
Electrospray ionization mass spectrometry (ESI MS) has emerged recently as a powerful tool for analyzing many structural and behavioral aspects of metalloproteins in great detail. In this review we discuss recent developments in the field, placing particular emphasis on the unique features of ESI MS that lend themselves to metalloprotein characterization at a variety of levels. Direct mass measurement enables the determination of protein-metal ion binding stoichiometry in solution and metalloprotein higher order structure in the case of multi-subunit proteins. MS techniques have been developed for determining the locations of metal-binding centers, metal oxidation states and reaction intermediates of metal-containing enzymes. Other ESI MS techniques are also discussed, such as protein ion charge state distributions and hydrogen/deuterium exchange studies, which can be used to measure metal binding affinities and to shed light on vital dynamic aspects of the functional properties of metalloproteins endowed by metal binding. 相似文献
Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion‐mobility mass spectrometry (IM‐MS) and hydrogen/deuterium exchange mass spectrometry (HDX‐MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins. 相似文献
The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (microESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/microESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D(28K). Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F. 相似文献