Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 461
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Artigo em Inglês | MEDLINE | ID: mdl-32004940

RESUMO

Flibanserin (FLB) is the first FDA approved drug showed to have significant activity against sexual desire disorder of premenopausal and postmenopausal women. Unfortunately, FLB is used as an adulterant in dietary supplement products as a performance enhancer in sports. Identification of FLB and its metabolites in the biological samples requires an authenticated analytical technique. The aim of this study was to identify N-oxide metabolite of FLB in microsomal and S9 human liver enzyme fractions, rat urine and feces. There are several N-oxide reported as genotoxic impurity or reactive metabolites based on position of N-oxide in piperazine ring. This study also describes the strategy to utilize degradation chemistry for isolation of N-oxide and its step-wise characterization. An LC-MS method has been developed and employed for identifying the N-oxide metabolite of FLB. The targeted N-oxide metabolite in the extracted ion chromatogram of the in vitro and in vivo samples has been confirmed by analyzing the changes in observed mass at m/z 407.1693. Major distinguished abundant ions at m/z 243.1104, 190.0974, 161.0705, 119.0601 confirmed the structure of the metabolite. This study will help to understand the oxidative potential of FLB in toxicokinetic study. The developed method can be useful to identify FLB or its N-oxide metabolite in dope testing in future. This is the first time to report a strategy to utilize degradation chemistry for N-oxide metabolite characterization. In this study, isolated N-oxidative degradation product was used to confirm N-oxide metabolite which was characterized by LC-MS through H/D exchange and structure was ensured by NMR spectroscopy (1H, COSY).


Assuntos
Benzimidazóis , Medição da Troca de Deutério/métodos , Fezes/química , Espectrometria de Massas em Tandem/métodos , Administração Oral , Animais , Benzimidazóis/administração & dosagem , Benzimidazóis/análise , Benzimidazóis/química , Benzimidazóis/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Espectroscopia de Ressonância Magnética , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley
2.
Anal Bioanal Chem ; 412(7): 1693-1700, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31993727

RESUMO

Major histocompatibility complex class I chain-related A and B (MICA/B) are cell-surface proteins that act as ligands to natural killer cell receptors, NKG2D, expressed on immune cells. Prevention of proteolytic shedding of MICA/B to retain their integrity on the cell surface has become a therapeutic strategy in immuno-oncology. Given the unique mechanism of MICA/B shedding, structural characterization of MICA/B and therapeutic agent interaction is important in the drug discovery process. In this study, we describe the practical utility of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in epitope mapping studies of a cohort of four monoclonal antibodies targeting MICA in a rapid manner. HDX-MS followed by electron-transfer dissociation allows high-resolution refinement of binding epitopes. This integrated strategy offers, for the first time, molecular-level understanding of MICA's conformational dynamics in solution as well as the unique mechanism of actions of these antibodies in targeting MICA. Graphical abstract.


Assuntos
Anticorpos Monoclonais/imunologia , Medição da Troca de Deutério/métodos , Mapeamento de Epitopos/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Espectrometria de Massas/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Transporte de Elétrons , Humanos
3.
J Am Soc Mass Spectrom ; 30(12): 2795-2804, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31720974

RESUMO

Mass spectrometry (MS)-based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.


Assuntos
Benzodiazepinas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Espectrometria de Massas em Tandem/métodos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Benzodiazepinas/química , Proteínas de Ciclo Celular/química , Medição da Troca de Deutério/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Radical Hidroxila/análise , Radical Hidroxila/metabolismo , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Fatores de Transcrição/química
4.
Nat Methods ; 16(7): 595-602, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31249422

RESUMO

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Dados , Concentração de Íons de Hidrogênio
5.
Rapid Commun Mass Spectrom ; 33(15): 1248-1257, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31034666

RESUMO

RATIONALE: Mass spectra processing in protein hydrogen/deuterium (H/D) exchange has been remarkably improved by the introduction of fitting of the amide exchange probabilities to peptide isotopic envelope intensities (Kan et al., 2013), in contrast to methods in which only the peptide deuterium uptakes (centroid shifts of isotopic envelopes) are used. However, the known implementations are based on the general fitting routines that use only the objective function values. Besides, applicability of more than one fitting method makes necessary their comparative evaluation. METHODS: Two fitting methods were considered: the common least squares and the fitting of the multinomial distribution representing the number of deuterium atoms exchanged in the individual peptides. Both methods were applied either directly to the isotopic envelope data or to the deuterium distributions obtained by envelope deconvolution (i.e. de-isotoping). RESULTS: An autonomous Matlab script was prepared, based on the exact expressions for the gradient and Hessian of the objective function, with the trust-region algorithm implemented in the compact analytical form recently made available. The least-squares fitting to the envelope data produced the best results, with the greatest precision and good coverage of exact values by the confidence intervals. The deuterium distributions were sensitive to the (simulated) experimental error whose progression by envelope deconvolution caused degradation in accuracy. The multinomial distribution fitting exhibited poor performance due to inadequate representation of the experimental error and missing of the appropriate weight parameters. Some specific peptide arrangement details were discussed as potential sources of ambiguity in the fitting results. CONCLUSIONS: The method of fitting to peptide isotopic envelopes has been improved by using the exact gradient and Hessian of the objective function. The fitting should be repeated with different initial guesses in order to find not only the global minimum, but also the local minima with similar depths which may exist due to eventual ambiguity of the fitting results.


Assuntos
Medição da Troca de Deutério/métodos , Peptídeos/química , Proteínas/química , Algoritmos , Amidas/química , Deutério/análise , Hidrogênio/análise , Espectrometria de Massas
6.
MAbs ; 11(4): 779-788, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30890021

RESUMO

High concentration biotherapeutic formulations are often required to deliver large doses of drugs to achieve a desired degree of efficacy and less frequent dose. However, highly concentrated protein-containing solutions may exhibit undesirable therapeutic properties, such as increased viscosity, aggregation, and phase separation that can affect drug efficacy and raise safety issues. The characterization of high concentration protein formulations is a critical yet challenging analytical task for therapeutic development efforts, due to the lack of technologies capable of making accurate measurements under such conditions. To address this issue, we developed a novel dilution-free hydrogen/deuterium exchange (HDX) mass spectrometry (MS) method for the direct conformational analysis of high concentration biotherapeutics. Here, we particularly focused on studying phase separation phenomenon that can occur at high protein concentrations. First, two aliquots of monoclonal antibodies (mAbs) were dialyzed in either hydrogen- or deuterium-containing buffers at low salt and pH. Phases that separated were then discretely sampled and subjected to dilution-free HDX-MS analysis through mixing the non-deuterated and deuterated protein aliquots. Our HDX-MS results analyzed at a global protein level reveal less deuterium incorporation for the protein-enriched phase compared to the protein-depleted phase present in high concentration formulations. A peptide level analysis further confirmed these observed differences, and a detailed statistical analysis provided direct information surrounding the details of the conformational changes observed. Based on our HDX-MS results, we propose possible structures for the self-associated mAbs present at high concentrations. Our new method can potentially provide useful insights into the unusual behavior of therapeutic proteins in high concentration formulations, aiding their development.


Assuntos
Anticorpos Monoclonais/química , Produtos Biológicos/química , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Deutério/química , Diálise , Humanos , Hidrogênio/química , Conformação Proteica
7.
Anal Chim Acta ; 1054: 114-121, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30712581

RESUMO

Biological therapeutics are established as major contributors to the pharmaceutical pipeline. Many of these biological drugs are lyophilized to preserve their conformation and reduce decomposition during storage and shipping. Therefore, understanding and controlling the effects of lyophilization on protein higher order structure is critical for commercialization of biologics. Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a well-established technique for studying protein higher order structure. Previous publications have demonstrated a solid state HDX (ssHDX) method for labeling formulated, lyophilized proteins to assess their physical stability during, but this process still suffered from low throughput and undesired back exchange. Recently, our group described a method combining HDX-MS with MALDI to greatly reduce the time of analysis and nearly eliminate H/D back-exchange, but that method was not suited for interrogating solid samples. This work integrates the two techniques to assess and predict the stability of peptides and proteins following mixing and lyophilization with various excipient formulations. Sample mixing and handling were performed through the use of a bench-top robotics and programmed data MALDI-MS acquisition allowed for monitoring deuterium incorporation for dried peptides and protein samples following continuous labeling with D2O vapor. Effects of excipients upon peptide stability were also tracked and compared to a control for a three day labeling time course. This workflow is automated and free from back-exchange. As demonstrated by deuterium retention of bradykinin, these features serve to reduce experimental error normally associated with conventional deuterium exchange experiments. The proposed union of MALDI-MS and ssHDX can be applied to study higher order structure of proteins and peptides and the effects of added excipients in an environment that closely resembles the storage and shipping conditions of biopharmaceuticals and may be beneficial in giving insights studying protein structural dynamics in solids.


Assuntos
Medição da Troca de Deutério/métodos , Peptídeos/química , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Automação , Liofilização , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Estabilidade Proteica
8.
J Biomol NMR ; 73(1-2): 43-48, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30661150

RESUMO

Protein molecules sample different conformations in solution and characterizing these conformations is crucial to understanding protein function. 15N CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a 'visible' major state and 'invisible' minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope effect on the amide 15N chemical shifts. However at moderately high temperatures (~ 50 °C) that are sometimes required to populate protein minor conformers to levels (~ 1%) that can be detected by CEST experiments solvent H/D exchange can lead to 'dips' in low B115N CEST profiles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of ~ 18 kDa T4 Lysozyme (T4L) at 50 °C and the ~ 11 kDa E. coli hibernation promoting factor (HPF) at 52 °C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D2O lock or by using a small amount (~ 1-3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer.


Assuntos
Medição da Troca de Deutério/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Amidas/química , Artefatos , Deutério , Proteínas de Escherichia coli/química , Muramidase/química , Isótopos de Nitrogênio , Proteínas Ribossômicas/química , Temperatura
9.
J Am Soc Mass Spectrom ; 30(2): 235-247, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30353291

RESUMO

The conformations of glycans are crucial for their biological functions. In-electrospray ionization (ESI) hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a promising technique for studying carbohydrate conformations since rapidly exchanging functional groups, e.g., hydroxyls, can be labeled on the timeframe of ESI. However, regular application of in-ESI HDX to characterize carbohydrates requires further analysis of the in-ESI HDX methodology. For instance, in this method, HDX occurs concurrently to the analyte transitioning from solution to gas-phase ions. Therefore, there is a possibility of sampling both gas-phase and solution-phase conformations of the analyte. Herein, we differentiate in-ESI HDX of metal-adducted carbohydrates from gas-phase HDX and illustrate that this method analyzes solvated species. We also systematically examine the effects of ESI parameters, including spray solvent composition, auxiliary gas flow rate, sheath gas flow rate, sample infusion rate, sample concentration, and spray voltage, and discuss their effects on in-ESI HDX. Further, we model the structural changes of a trisaccharide, melezitose, and its intramolecular and intermolecular hydrogen bonding in solvents with different compositions of methanol and water. These molecular dynamic simulations support our experimental results and illustrate how an individual ESI parameter can alter the conformations we sample by in-ESI HDX. In total, this work illustrates how the fundamental processes of ESI alter the magnitude of HDX for carbohydrates and suggest parameters that should be considered and/or optimized prior to performing experiments with this in-ESI HDX technique. Graphical Abstract ᅟ.


Assuntos
Carboidratos/química , Medição da Troca de Deutério/métodos , Metais/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Carboidratos/análise , Gases/química , Metanol/química , Simulação de Dinâmica Molecular , Solventes/química , Trissacarídeos/análise , Trissacarídeos/química
10.
Methods Mol Biol ; 1873: 53-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30341603

RESUMO

Cystic fibrosis (CF) is one of the most common, lethal autosomal recessive diseases in Caucasians with a life expectancy of 37-47 years. The CF transmembrane conductance regulator (CFTR) is a plasma membrane ion channel, confined to apical membrane of epithelia, and ensures transepithelial water and solute movement across secretory epithelia in several organs. Numerous CF mutations, including the most prevalent deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) leads to CFTR global misfolding and premature intracellular degradation at the endoplasmic reticulum (ER). To better understand the misfolding mechanism caused by CF-causing point mutations in the NBD1, which is poorly understood, differential scanning fluorimetry (DSF) and hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) are the choice of techniques. These established methods can measure the conformational dynamics of the NBD1 globally and at peptide resolution level by monitoring backbone amide HDX, respectively, and will be instrumental to evaluate the mechanism of action of CF mutations and folding correctors that rescue CFTR folding defects via stabilizing the mutant NBD1.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Medição da Troca de Deutério/métodos , Fluorometria/métodos , Espectrometria de Massas/métodos , Simulação de Dinâmica Molecular , Mutação Puntual , Sequência de Aminoácidos , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
J Am Soc Mass Spectrom ; 30(1): 45-57, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30460642

RESUMO

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND3-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Complexos Multiproteicos/análise , Complexos Multiproteicos/química , Animais , Bovinos , Citocromos c/análise , Citocromos c/química , Citocromos c/metabolismo , Medição da Troca de Deutério/instrumentação , Gases/química , Humanos , Lactalbumina/análise , Lactalbumina/química , Lactalbumina/metabolismo , Espectrometria de Massas/instrumentação , Complexos Multiproteicos/metabolismo , Pré-Albumina/análise , Pré-Albumina/química , Pré-Albumina/metabolismo , Multimerização Proteica
12.
J Am Soc Mass Spectrom ; 30(1): 58-66, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30280315

RESUMO

Hydrogen deuterium exchange mass spectrometry (HDX-MS) has significant potential for protein structure initiatives but its relationship with protein conformations is unclear. We report on the efficacy of HDX-MS to distinguish between native and non-native proteins using a popular approach to calculate HDX protection factors (PFs) from protein structures. The ability of HDX-MS to identify native protein conformations is quantified by binary structural classification such that merits of the approach for protein modelling can be quantified and better understood. We show that highly accurate PF calculations are not a prerequisite for HDX-MS simulations that are capable of effectively discriminating between native and non-native protein folds. The simulations can also be performed directly on unique structures facilitating high-throughput evaluation of many alternate conformations. The ability of HDX-MS to classify the conformations of homo-protein assemblies is also investigated. In contrast to protein monomers, we show a significant lack of correspondence between the simulated and experimental HDX-MS data for these systems with a subsequent decrease in the ability of HDX-MS to identify native states. However, we demonstrate surprisingly high diagnostic ability of the simulated data for assemblies in which a significant proportion of the individual chains occupy protein-protein interfaces. We relate this to the number of peptides that can sample alternate subunit orientations and discuss these observations within the larger context of applying HDX-MS to evaluate protein structures. Graphical Abstract.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Dobramento de Proteína , Proteínas/química , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Lactalbumina/análise , Lactalbumina/química , Modelos Moleculares , Proteínas/análise , Ribonucleases/análise , Ribonucleases/química , Fluxo de Trabalho
13.
Protein Pept Lett ; 26(1): 16-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30543159

RESUMO

BACKGROUND: Structural biology has provided a fundamental understanding of protein structure and mechanistic insight into their function. However, high-resolution structures alone are insufficient for a complete understanding of protein behavior. Higher energy conformations, conformational changes, and subtle structural fluctuations that underlie the proper function of proteins are often difficult to probe using traditional structural approaches. Hydrogen/Deuterium Exchange with Mass Spectrometry (HDX-MS) provides a way to probe the accessibility of backbone amide protons under native conditions, which reports on local structural dynamics of solution protein structure that can be used to track complex structural rearrangements that occur in the course of a protein's function. CONCLUSION: In the last 20 years the advances in labeling techniques, sample preparation, instrumentation, and data analysis have enabled HDX to gain insights into very complex biological systems. Analysis of challenging targets such as membrane protein complexes is now feasible and the field is paving the way to the analysis of more and more complex systems.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Conformação Proteica , Proteínas/química
14.
Pharm Res ; 36(1): 24, 2018 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-30536043

RESUMO

PURPOSE: Tryptophan's (Trp) unique hydrophobic and structural properties make it an important antigen binding motif when positioned in complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs). Oxidation of Trp residues within the CDR can deleteriously impact antigen binding, particularly if the CDR conformation is altered. The goal of this study was to evaluate the conformational and functional impact of Trp oxidation for two mAb subtypes, which is essential in determining the structure-function relationship and establishing appropriate analytical control strategies during protein therapeutics development. METHODS: Selective Trp oxidation was induced by 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) treatment in the presence of free methionine (Met). The native and chemically oxidized mAbs were characterized by hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes and surface plasmon resonance (SPR) for antigen-antibody binding. RESULTS: Treatment of mAbs with AAPH selectively oxidized solvent accessible Trp residues. Oxidation of Trp within or in proximity of CDRs increased conformational flexibility in variable domains and disrupted antigen binding. CONCLUSIONS: Trp oxidation in CDRs can adversely impact mAbs' conformation and antigen binding. Trp oxidation should be carefully evaluated as part of critical quality attribute assessments. Oxidation susceptible Trp should be closely monitored during process development for mAbs to establish appropriate analytical control for manufacturing of drug substance and drug product.


Assuntos
Anticorpos Monoclonais/química , Regiões Determinantes de Complementaridade/química , Deutério/química , Hidrogênio/química , Triptofano/química , Antígenos/química , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Oxirredução , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície/métodos
15.
Nat Commun ; 9(1): 5366, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560918

RESUMO

Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N1-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.


Assuntos
Autoimunidade/genética , Proteína DEAD-box 58/genética , Imunidade Inata/genética , Capuzes de RNA/imunologia , RNA de Cadeia Dupla/imunologia , Adenosina Trifosfatases/metabolismo , Domínio de Ativação e Recrutamento de Caspases/imunologia , Proteína DEAD-box 58/química , Proteína DEAD-box 58/imunologia , Proteína DEAD-box 58/metabolismo , Medição da Troca de Deutério/métodos , Mutação com Ganho de Função , Guanosina/análogos & derivados , Guanosina/química , Guanosina/imunologia , Guanosina/metabolismo , Helicase IFIH1 Induzida por Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Espectrometria de Massas/métodos , Metilação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Ligação Proteica/imunologia , Capuzes de RNA/química , Capuzes de RNA/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA Viral/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
17.
J Am Soc Mass Spectrom ; 29(12): 2402-2412, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30324261

RESUMO

The dominant gas-phase conformer of [M+3H]3+ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. The [M+3H]3+ peptide ions are observed predominantly as a relatively compact conformer type. Upon subjecting these ions to electron transfer dissociation (ETD), the level of protection for each amino acid residue in the peptide sequence is assessed. The overall per-residue deuterium uptake is observed to be relatively more efficient for the neutral residues than for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison, the N-terminal and C-terminal regions of the serine peptide show greater relative protection compared with interior residues. Molecular dynamics (MD) simulations have been used to generate candidate structures for collision cross section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for select structural candidates from MD simulations has been used to suggest conformer types that could contribute to the observed HDX patterns. The results are discussed with respect to recent studies employing extensive MD simulations of gas-phase structure establishment of a peptide system. Graphical Abstract ᅟ.


Assuntos
Medição da Troca de Deutério/métodos , Peptídeos/química , Conformação Proteica em alfa-Hélice , Espectrometria de Massas em Tandem/métodos , Íons/química , Simulação de Dinâmica Molecular
18.
J Phys Chem B ; 122(49): 11206-11217, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30179470

RESUMO

Solvent exchange rates provide important information about the structural and dynamical properties of biomolecules. A large number of NMR experiments have been developed to measure such rates in proteins, the great majority of which quantify the buildup of signals from backbone amides after initial perturbation of water magnetization. Here we present a different approach that circumvents the main limitations that result from these classical hydrogen exchange NMR experiments. Building on recent developments that enable rapid recording of chemical exchange saturation transfer (CEST) pseudo-3D data sets, we describe a 15N-based CEST scheme for measurement of solvent exchange in proteins that exploits the one-bond 15N deuterium isotope shift. The utility of the approach is verified with an application to a 236 residue intrinsically disordered protein domain under conditions where it phase separates and a second application involving a mutated form of the domain that does not phase separate, establishing very similar hydrogen exchange rates for both samples. The methodology is well suited for studies of hydrogen exchange in any 15N-labeled biomolecule. A discussion of the merits of the CEST experiment in relation to the popular CLEANEX-PM scheme is presented.


Assuntos
RNA Helicases DEAD-box/química , Deutério/química , Fragmentos de Peptídeos/química , Amidas/química , RNA Helicases DEAD-box/genética , Medição da Troca de Deutério/métodos , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Mutação , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/genética , Domínios Proteicos , Solventes/química
19.
J Proteome Res ; 17(11): 3614-3627, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30222357

RESUMO

Over the past decade, a suite of new mass-spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein-ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL-MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states.


Assuntos
Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteoma/química , Proteômica/tendências , Animais , Reagentes para Ligações Cruzadas/química , Bases de Dados de Proteínas , Medição da Troca de Deutério/métodos , Humanos , Marcação por Isótopo/métodos , Ligantes , Espectrometria de Massas/instrumentação , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Proteínas/metabolismo , Proteínas/ultraestrutura , Proteólise , Proteoma/ultraestrutura , Proteômica/instrumentação , Proteômica/métodos , Análise de Sequência de Proteína/instrumentação , Análise de Sequência de Proteína/métodos , Análise de Sequência de Proteína/estatística & dados numéricos , Termodinâmica
20.
J Am Soc Mass Spectrom ; 29(12): 2413-2426, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30267362

RESUMO

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 ß2-microglobulin (ß2m) 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. Graphical Abstract ᅟ.


Assuntos
Medição da Troca de Deutério/métodos , Microglobulina beta-2/química , Humanos , Simulação de Dinâmica Molecular , Oxirredução , Fragmentos de Peptídeos/química , Processos Fotoquímicos , Proteínas Recombinantes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA