RESUMEN
A heteroleptic [Pd2L2L'2]4+ coordination cage containing a photoswitchable azobenzene-derived ligand catalyzes the Michael addition reaction between methyl vinyl ketone and benzoyl nitromethane within its cavity. The corresponding homoleptic cages are catalytically inactive. The heteroleptic cage can be reversibly disassembled and reassembled using 530 and 405 nm light, respectively, allowing catalysis within the cage to be switched OFF and ON at will.
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Per- and polyfluoroalkyl substances (PFAS) are known for their high environmental persistence and potential toxicity. The presence of PFAS has been reported in many dairy products. However, the mechanisms underlying the accumulation of PFAS in these products remain unclear. Here, we used native mass spectrometry and molecular dynamics simulations to probe the interactions between 19 PFAS of environmental concern and two isoforms of the major bovine whey protein ß-lactoglobulin (ß-LG). We observed that six of these PFAS bound to both protein isoforms with low- to mid-micromolar dissociation constants. Based on quantitative, competitive binding experiments with endogenous ligands, PFAS can bind orthosterically and preferentially to ß-LG's hydrophobic ligand-binding calyx. ß-Cyclodextrin can also suppress binding of PFAS to ß-LG owing to the ability of ß-cyclodextrin to directly sequester PFAS from solution. This research sheds light on PFAS-ß-LG binding, suggesting that such interactions could impact lipid-fatty acid transport in bovine mammary glands at high PFAS concentrations. Furthermore, our results highlight the potential use of ß-cyclodextrin in mitigating PFAS binding, providing insights toward the development of strategies to reduce PFAS accumulation in dairy products and other biological systems.
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Fluorocarburos , Lactoglobulinas , Leche , Animales , Lactoglobulinas/metabolismo , Lactoglobulinas/química , Bovinos , Leche/química , Leche/metabolismo , Fluorocarburos/química , Fluorocarburos/metabolismo , Simulación de Dinámica Molecular , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismo , Sitios de Unión , Unión ProteicaRESUMEN
Small molecule drug discovery has been propelled by the continual development of novel scientific methodologies to occasion therapeutic advances. Although established biophysical methods can be used to obtain information regarding the molecular mechanisms underlying drug action, these approaches are often inefficient, low throughput, and ineffective in the analysis of heterogeneous systems including dynamic oligomeric assemblies and proteins that have undergone extensive post-translational modification. Native mass spectrometry can be used to probe protein-small molecule interactions with unprecedented speed and sensitivity, providing unique insights into polydisperse biomolecular systems that are commonly encountered during the drug discovery process. In this review, we describe potential and proven applications of native MS in the study of interactions between small, drug-like molecules and proteins, including large multiprotein complexes and membrane proteins. Approaches to quantify the thermodynamic and kinetic properties of ligand binding are discussed, alongside a summary of gas-phase ion activation techniques that have been used to interrogate the structure of protein-small molecule complexes. We additionally highlight some of the key areas in modern drug design for which native mass spectrometry has elicited significant advances. Future developments and applications of native mass spectrometry in drug discovery workflows are identified, including potential pathways toward studying protein-small molecule interactions on a whole-proteome scale.
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Descubrimiento de Drogas , Proteoma , Descubrimiento de Drogas/métodos , Espectrometría de Masas/métodos , Proteínas de la Membrana , TermodinámicaRESUMEN
Molecular glues stabilize interactions between E3 ligases and novel substrates to promote substrate degradation, thereby facilitating the inhibition of traditionally "undruggable" protein targets. However, most known molecular glues have been discovered fortuitously or are based on well-established chemical scaffolds. Efficient approaches for discovering and characterizing the effects of molecular glues on protein interactions are required to accelerate the discovery of novel agents. Here, we demonstrate that native mass spectrometry and mass photometry can provide unique insights into the physical mechanism of molecular glues, revealing previously unknown effects of such small molecules on the oligomeric organization of E3 ligases. When compared to well-established solution phase assays, native mass spectrometry provides accurate quantitative descriptions of molecular glue potency and efficacy while also enabling the binding specificity of E3 ligases to be determined in a single, rapid measurement. Such mechanistic insights should accelerate the rational development of molecular glues to afford powerful therapeutic agents.
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Fotometría , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Espectrometría de Masas , ProteolisisRESUMEN
Carbonic anhydrases (CAs) are a metalloenzyme family that have important roles in cellular processes including pH homeostasis and have been implicated in multiple pathological conditions. Small molecule inhibitors have been developed to target carbonic anhydrases, but the effects of post-translational modifications (PTMs) on the activity and inhibition profiles of these enzymes remain unclear. Here, we investigate the effects of phosphorylation, the most prevalent carbonic anhydrase PTM, on the activities and drug-binding affinities of human CAI and CAII, two heavily modified active isozymes. Using serine to glutamic acid (S > E) mutations to mimic the effect of phosphorylation, we demonstrate that phosphomimics at a single site can significantly increase or decrease the catalytic efficiencies of CAs, depending on both the position of the modification and the CA isoform. We also show that the S > E mutation at Ser50 of hCAII decreases the binding affinities of hCAII with well-characterized sulphonamide inhibitors including by over 800-fold for acetazolamide. Our findings suggest that CA phosphorylation may serve as a regulatory mechanism for enzymatic activity, and affect the binding affinity and specificity of small, drug and drug-like molecules. This work should motivate future studies examining the PTM-modification forms of CAs and their distributions, which should provide insights into CA physiopathological functions and facilitate the development of 'modform-specific' carbonic anhydrase inhibitors.
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Anhidrasas Carbónicas , Humanos , Anhidrasas Carbónicas/metabolismo , Anhidrasa Carbónica II , Fosforilación , Dominio Catalítico , Inhibidores de Anhidrasa Carbónica/química , Anhidrasa Carbónica IX/metabolismoRESUMEN
Fungal infection of grape berries (Vitis vinifera) by Botrytis cinerea frequently coincides with harvest, impacting both the yield and quality of grape and wine products. A rapid and non-destructive method for identifying B. cinerea infection in grapes at an early stage prior to harvest is critical to manage loss. In this study, zeolitic imidazolate framework-8 (ZIF-8) crystal was applied as an absorbent material for volatile extraction from B. cinerea infected and healthy grapes in a vineyard, followed by thermal desorption gas chromatography-mass spectrometry. The performance of ZIF-8 in regard to absorbing and trapping the targeted volatiles was evaluated with a standard solution of compounds and with a whole bunch of grapes enclosed in a glass container to maintain standard sampling conditions. The results from the sampling methods were then correlated to B. cinerea infection in grapes, as measured and determined by genus-specific antigen quantification. Trace levels of targeted compounds reported as markers of grape B. cinerea infection were successfully detected with in-field sampling. The peak area counts for volatiles 3-octanone, 1-octen-3-one, 3-octanol, and 1-octen-3-ol extracted using ZIF-8 were significantly higher than values achieved using Tenax®-TA from field testing and demonstrated good correlation with B. cinerea infection severities determined by B. cinerea antigen detection.
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Vitis , Vino , Vitis/química , Vino/análisis , Botrytis , Cromatografía de Gases y Espectrometría de Masas/métodos , Frutas/químicaRESUMEN
Theta capillary nanoelectrospray ionization (θ-nanoESI) can be used to "supercharge" protein ions directly from solution for detection by mass spectrometry (MS). In native top-down MS, the extent of protein charging is low. Given that ions with more charge fragment more readily, increasing charge can enhance the extent of sequence information obtained by top-down MS. For θ-nanoESI, dual-channeled nanoESI emitters are used to mix two solutions in low to sub-µs prior to MS. The mechanism for θ-nanoESI mixing has been reported to primarily occur: (i) in a single shared Taylor cone and in the droplets formed from the Taylor cone or (ii) by the fusion of droplets formed from two separate Taylor cones. Using θ-nanoESI-ion mobility MS, native protein solutions were rapidly mixed with denaturing supercharging solutions to form protein ions in significantly higher charge states and with more elongated structures than those formed by premixing the solutions prior to nanoESI-MS. If θ-nanoESI mixing occurred in the Taylor cone and in the droplets resulting from the single Taylor cone, then the extent of protein charging and unfolding should be comparable to or less than that obtained by premixing solutions. Thus, these data are consistent with mixing occurring via droplet fusion rather than in the Taylor cone prior to ESI droplet formation. These data also suggest that highly charged protein ions can be formed by the near-complete mixing of each solution. The presence of supercharging additives in premixed solutions can suppress volatile electrolyte evaporation, limiting the extent of protein charging compared to when the additive is delivered via one channel of a θ-nanoESI emitter. In θ-nanoESI, the formation of two Taylor cones can presumably result in substantial electrolyte evaporation from the ESI droplets containing native-like proteins prior to droplet fusion, thereby enhancing ion charging.
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Proteínas , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Movilidad Iónica , Iones , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
A photoswitchable ligand and palladium(II) ions form a dynamic mixture of self-assembled metallosupramolecular structures. The photoswitching ligand is an ortho-fluoroazobenzene with appended pyridyl groups. Combining the E-isomer with palladium(II) salts affords a double-walled triangle with composition [Pd3 L6 ]6+ and a distorted tetrahedron [Pd4 L8 ]8+ (1 : 2 ratio at 298â K). Irradiation with 410â nm light generates a photostationary state with approximately 80 % of the E-isomer of the ligand and results in the selective disassembly of the tetrahedron, the more thermodynamically stable structure, and the formation of the triangle, the more kinetically inert product. The triangle is then slowly transformed back into the tetrahedron over 2â days at 333â K. The Z-isomer of the ligand does not form any well-defined structures and has a thermal half-life of 25â days at 298â K. This approach shows how a thermodynamically preferred self-assembled structure can be reversibly pumped to a kinetic trap by small perturbations of the isomer distribution using non-destructive visible light.
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Differential ion mobility (DMS) is a versatile ion separation method that is often integrated with mass spectrometry (MS). In DMS, extremely high electric fields are used such that ion mobility depends non-linearly on electric field and thus, ion separations can be more orthogonal to MS than lower field ion mobility-based methods. DMS can have sufficiently high resolution to be used for enantiomer analysis of small molecules and to separate protein ions with peak widths comparable to those obtained for peptides. However, the performance of high resolution DMS-MS can be limited owing to the substantial loss of ions (>10-fold) that can occur upon their transfer from atmospheric pressure (where DMS separation typically occurs) to vacuum through a narrow conductance limited inlet (e.g. capillary) to the MS. Here, results from simulated ion trajectory simulations suggest that in high resolution DMS most ions can be lost by 'crashing' onto the narrow capillary inlet after exiting the DMS separation channel. To enhance DMS sensitivity and resolving power, an integrated DMS-MS interface concept is reported that consists of a slit electrode and a 12-electrode atmospheric pressure ion funnel (APIF). By using an APIF with slit entrance, the simulated ion transmission efficiencies increase by up to 257% for singly charged ions ([DMMP + H]+, [tryptophan + H]+, and [(2-dodecanone)2 + H]+) and by 209% for [ubiquitin + 12H]12+, without compromising resolving power. The use of APIF improves the ion focussing from the DMS exit to the MS capillary to improve sensitivity, and the slit ensures that ion dispersion in the analytically relevant direction perpendicular to the DMS electrodes is restricted to enhance resolution. By narrowing the slit of the DMS-Slit-APIF interface, the DMS resolving power can be increased further by at least 20%. Overall, these results indicate that the integrated DMS-Slit-APIF interface is promising for improving the sensitivity and resolution for many different types of DMS-MS experiments.
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Presión Atmosférica , Espectrometría de Movilidad Iónica , Iones , Espectrometría de Masas , EstereoisomerismoRESUMEN
A photoswitchable ligand based on azobenzene is self-assembled with palladium(II) ions to form a [Pd2 (E-L)4 ]4+ cage. Irradiation with 470â nm light results in the near-quantitative switching to a monomeric species [Pd(Z-L)2 ]2+ , which can be reversed by irradiation with 405â nm light, or heat. The photoswitching selectivity towards the metastable isomer is significantly improved upon self-assembly, and the thermal half-life is extended from 40â days to 850â days, a promising approach for tuning photoswitching properties.
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The structural diversity of natural products offers unique opportunities for drug discovery, but challenges associated with their isolation and screening can hinder the identification of drug-like molecules from complex natural product extracts. Here we introduce a mass spectrometry-based approach that integrates untargeted metabolomics with multistage, high-resolution native mass spectrometry to rapidly identify natural products that bind to therapeutically relevant protein targets. By directly screening crude natural product extracts containing thousands of drug-like small molecules using a single, rapid measurement, we could identify novel natural product ligands of human drug targets without fractionation. This method should significantly increase the efficiency of target-based natural product drug discovery workflows.
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Productos Biológicos/química , Ligandos , Proteínas/química , Productos Biológicos/metabolismo , Anhidrasa Carbónica I/química , Anhidrasa Carbónica I/metabolismo , Cromatografía Líquida de Alta Presión , Humanos , Metabolómica/métodos , Proteínas/metabolismo , Espectrometría de Masas en TándemRESUMEN
Ultraviolet photodissociation (UVPD) is a powerful and rapidly developing method in top-down proteomics. Sequence coverages can exceed those obtained with collision- and electron-induced fragmentation methods. Because of the recent interest in UVPD, factors that influence protein fragmentation and sequence coverage are actively debated in the literature. Here, we performed top-down 213 nm UVPD experiments on a 7 T Fourier-transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for the model proteins ubiquitin, myoglobin and cytochrome c that were electrosprayed from native, denaturing and supercharging solutions in order to investigate the effect of protein charge states on UVPD fragments. By performing UVPD in ultrahigh vacuum, factors associated with collisional cooling and any ion activation during transfer between mass analyzers can be largely eliminated. Sequence coverage increased from <10% for low charge states to >60% for high charge states for all three proteins. This trend is influenced by the overall charge state, i.e., charges per number of amino acid residues, and to a lesser degree by associated structural changes of protein ions of different charge states based on comparisons to published collision-cross section measurements. To rationalize this finding, and correlate sequence ion formation and identity with the number and location of protons, UVPD results were compared to protonation sites predicted based on electrostatic modelling. Assuming confined protonation sites, these results indicate the presence of two general fragmentation types; i.e., charge remote and charge directed. For moderately high protein charge states, fragment ions mostly originate in regions between likely protonation sites (charge remote), whereas sequence ions of highly charge protein ions occur either near backbone amide protonation sites at low-basicity residues (charge directed) or at charge remote sites (i.e., high-basicity residues). Overall, our results suggest that top-down 213 UVPD performance in the zero-pressure limit depends strongly on protein charge states and protonation sites can influence the location of backbone cleavages.
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Proteómica , Rayos Ultravioleta , Iones , Espectrometría de Masas , ProtonesRESUMEN
Inorganic anions inhibit the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) generally by coordinating to the active site metal ion. Cyanate was reported as a non-coordinating CA inhibitor but those erroneous results were subsequently corrected by another group. We review the anion CA inhibitors (CAIs) in the more general context of drug design studies and the discovery of a large number of inhibitor classes and inhibition mechanisms, including zinc binders (sulphonamides and isosteres, dithiocabamates and isosteres, thiols, selenols, benzoxaboroles, ninhydrins, etc.); inhibitors anchoring to the zinc-coordinated water molecule (phenols, polyamines, sulfocoumarins, thioxocoumarins, catechols); CAIs occluding the entrance to the active site (coumarins and derivatives, lacosamide), as well as compounds that bind outside the active site. All these new chemotypes integrated with a general procedure for obtaining isoform-selective compounds (the tail approach) has resulted, through the guidance of rigorous X-ray crystallography experiments, in the development of highly selective CAIs for all human CA isoforms with many pharmacological applications.
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Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Complejos de Coordinación/farmacología , Diseño de Fármacos , Zinc/farmacología , Aniones/síntesis química , Aniones/química , Aniones/farmacología , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Cristalografía por Rayos X , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Modelos Moleculares , Zinc/químicaRESUMEN
In mass spectrometry (MS), a major loss of ions can readily occur during their transfer from atmospheric pressure to a lower pressure, which limits performance. Here, we report an ion funnel that can be used to effectively focus ions at ambient pressure (â¼777 Torr) to significantly enhance performance in electrospray ionization (ESI) MS. For seven singly charged test ions (m/z 124-1131), the ambient pressure ion funnel (APIF) is demonstrated to improve ion abundances, sensitivity, and detection limits by up to factors of â¼17, â¼16, and â¼3, respectively, compared to the operation of conventional ESI-MS. Simulated ion trajectories were used to rationalize the enhanced performance of the APIF, which is attributed primarily to using a relatively high RF field amplitude to radially confine ions, a high DC field, and a wide exit ring electrode. The effective focusing of ions at ambient pressures should be beneficial in the future for improving the performance of (i) additional methods that ionize molecules at atmospheric pressure, (ii) ambient pressure ion mobility-based instruments, and (iii) high flow rate liquid chromatography mass spectrometry platforms.
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We report the development of metal-organic framework (MOF)-based probes for the direct and rapid detection and quantification of perfluorooctanoic acid (PFOA) by mass spectrometry. Four water-resistant MOFs-ZIF-8, UiO-66, MIL88-A, and Tb2(BDC)3-were coated on poly(dopamine) precoated stainless steel needles and used to rapidly preconcentrate PFOA from water for direct analysis by nanoelectrospray ionization mass spectrometry. The analytical performance of each MOF for detecting PFOA was correlated with both the calculated binding energy of the MOF for PFOA and the relative change in the surface area of the MOF upon exposure to PFOA. MOF-functionalized probes can be used for the rapid (<5 min) and sensitive quantification of PFOA molecules at low ng L-1 levels in environmental water samples (i.e., tap water, rainwater, and seawater) with no sample preparation. The limit of detection of PFOA in ultrapure water was 11.0 ng L-1. Comparable accuracy to an accredited analytical method was achieved, despite the MOF-functionalized probe approach being â¼40 times quicker and requiring â¼10 times less sample. These features indicate that MOF-coated probes are promising for the direct and rapid monitoring of polyfluorinated substances and other pollutants in the field.
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Perfluoroalkyl substances (PFASs) persist and are ubiquitous in the environment. The origins of PFAS toxicity and how they specifically affect the functions of proteins remain unclear. Herein, we report that PFASs can strongly inhibit the activity of human carbonic anhydrases (hCAs), which are ubiquitous enzymes that catalyze the hydration of CO2, are abundant in the blood and organs of mammals, and involved in pH regulation, ion homeostasis, and biosynthesis. The interactions between PFASs and hCAs were investigated using stopped-flow kinetic enzyme-inhibition measurements, native mass spectrometry (MS), and ligand-docking simulations. Narrow-bore emitters in native MS with inner diameters of â¼300 nm were used to directly and simultaneously measure the dissociation constants of 11 PFASs to an enzyme, which was not possible using conventional emitters. The data from native MS and stopped-flow measurements were in excellent agreement. Of 15 PFASs investigated, eight can inhibit at least one of four hCA isozymes (I, II, IX, and XII) with submicromolar inhibition constants, including perfluorooctanoic acid, perfluorooctanesulfonamide, and perfluorooctanesulfonic acid. Some PFASs, including those with both short and long perfluoromethylene chains, can effectively inhibit at least one hCA isozyme with low nanomolar inhibition constants.
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Ácidos Alcanesulfónicos/farmacología , Caprilatos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Contaminantes Ambientales/farmacología , Fluorocarburos/farmacología , Ácidos Alcanesulfónicos/análisis , Caprilatos/análisis , Inhibidores de Anhidrasa Carbónica/análisis , Cristalografía por Rayos X , Contaminantes Ambientales/análisis , Fluorocarburos/análisis , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Ligandos , Espectrometría de Masas , Modelos Moleculares , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Netropsin is one of the first ligands to be discovered that selectively binds to the minor groove of DNA and is actively used as a scaffold for developing potential anticancer and antibiotic agents. The mechanism by which netropsin binds to hairpin DNA remains controversial with two competing mechanisms having been proposed. In one mechanism, netropsin binding induces a hairpin-to-duplex DNA transition. Alternatively, netropsin binds in two thermodynamically different modes at a single duplexed AATT site. Here, results from native mass spectrometry (MS) with nanoscale ion emitters indicate that netropsin can simultaneously and sequentially bind to both hairpin and duplex DNA. Duplex DNA was not detected using conventional MS with larger emitters because nanoscale emitters significantly reduce the extent of salt adduction to ligand-DNA complex ions, including in the presence of relatively high concentrations of nonvolatile salts. Based on native MS and polyacrylamide gel electrophoresis results, the abundances of hairpin and duplex DNA are unaffected by the addition of netropsin. By native MS, the binding affinities for five ligand-DNA and DNA-DNA interactions can be rapidly obtained simultaneously. This research indicates a "simultaneous binding mechanism" for the interactions of netropsin with DNA.
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ADN/metabolismo , Netropsina/metabolismo , ADN/genética , Electroforesis en Gel de Poliacrilamida , Secuencias Invertidas Repetidas , Unión Proteica , Espectrometría de Masa por Ionización de Electrospray/métodos , Streptomyces/químicaRESUMEN
Dielectric barrier discharge ionization (DBDI) is an emerging technique for ionizing volatile molecules directly from complex mixtures for sensitive detection by mass spectrometry (MS). In conventional DBDI, a high frequency and high voltage waveform with pulse widths of â¼50 µs (and â¼50 µs between pulses) is applied across a dielectric barrier and a gas to generate "low temperature plasma." Although such a source has the advantages of being compact, economical, robust, and sensitive, background ions from the ambient environment can be formed in high abundances, which limits performance. Here, we demonstrate that high voltage pulse widths as narrow as 100 ns with a pulse-to-pulse delay of â¼900 µs can significantly reduce background chemical noise and increase ion signal. Compared to microsecond pulses, â¼800 ns pulses can be used to increase the signal-to-noise and signal-to-background chemical noise ratios in DBDI-MS by up to 172% and 1300% for six analytes, including dimethyl methylphosphonate (DMMP), 3-octanone, and perfluorooctanoic acid. Using nanosecond pulses, the detection limit for DMMP and PFOA in human blood plasma can be lowered by more than a factor of 2 in comparison to microsecond pulses. In "nanopulsed" plasma ionization, the extent of internal energy deposition is as low as or lower than in electrospray ionization and micropulsed plasma ionization based on thermometer ion measurements. Overall, nanosecond high-voltage pulsing can be used to significantly improve the performance of DBDI-MS and potentially other ion sources involving high voltage waveforms.
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Organophosphates (OPs) are used worldwide as pesticides. However, acute and chronic exposure to OPs can cause serious adverse health effects. The mechanism of delayed OP toxicity is thought to involve off-target inhibition of serine proteases, although the precise molecular details remain unclear owing to the lack of an analytical method for global detection of protein targets of OPs. Here, we report the development of a mass spectrometry method to identify OP-adducted proteins from complex mixtures in a nontargeted manner. Human plasma was incubated with the OP dichlorvos that was 50% isotopically labeled and 50% unlabeled. Proteins and protein adducts were extracted, digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect "twin ions" of peptides that were covalently modified by a chemical reaction with dichlorvos. The LC-MS/MS data were processed by a blended data analytics software (Xenophile) to detect the amino acid residue sites of proteins that were covalently modified by exposure to OPs. We discovered that OPs can transmethylate the N, S, and O side chains of His, Cys, Glu, Asp, and Lys residues. For model systems, such transmethylation reactions were confirmed by LC-MS, nuclear magnetic resonance (NMR), and rationalized using electronic structure calculations. Methylation of the ubiquitous antioxidant glutathione by dichlorvos can decrease the reducing/oxidizing equilibrium of glutathione in liver extracts, which has been implicated in diseases and pathological conditions associated with delayed OP toxicity.
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Proteínas Sanguíneas/química , Nitrógeno/química , Organofosfatos/química , Oxígeno/química , Azufre/química , Cromatografía Liquida , Humanos , Metilación , Organofosfatos/toxicidad , Espectrometría de Masas en TándemRESUMEN
A ß-carbonic anhydrase (CA, EC 4.2.1.1) from the widespread bacterium Escherichia coli (EcoCAß), encoded by the CynT2 gene, has been investigated for its catalytic properties and enzymatic activation by a panel of amino acids and amines. EcoCAß showed a significant catalytic activity for the hydration of CO2 to bicarbonate and a proton, with a kinetic constant kcat of 5.3 × 105 s- and a Michaelis-Menten constant KM of 12.9 mM. The most effective EcoCAß activators were L- and D-DOPA, L-Tyr, 4-amino-Phe, serotonin and L-adrenaline, with KAs from 2.76 to 10.7 µM. L-His, 2-pyridyl-methylamine, L-Asn and L-Gln were relatively weak activators (KAs from 36.0 to 49.5 µM). D-His, L- and D-Phe, L- and D-Trp, D-Tyr, histamine, dopamine, 2-(aminoethyl)pyridine/piperazine/morpholine, L-Asp, L- and D-Glu have KAs from 11.3 to 23.7 µM. Endogenous CA activators may play a role in bacterial virulence and colonisation of the host.