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Contrary to the common but potentially misleading belief that when a protonated molecule is excited, it is its most stable protomer that will mandatorily dissociate, we demonstrate herein that, when rationalizing or predicting the chemistry of such ions, we should always search for the most labile protomer. This "most labile protomer" rule, based on the mobile proton model, states therefore that when a protonated molecule is heated, during ionization or by collisions for instance, the loosely bonded proton (H+ ) can acquire enough energy to detach itself from the most basic site of the molecule and then freely "walk through" the molecular framework to eventually find, if available, another protonation site, forming other less stable but more labile protomers, that is, protomers that may display lower dissociation thresholds. To demonstrate the validity of the "most labile protomer" rule as well as the misleading nature of the "most stable protomer" rule, we have selected several illustrative molecules and have collected their ESI(+)-MS/MS. To compare energies of precursors and products, we have also performed PM7 calculations and elaborated potential energy surface diagrams for their possible protomers and dissociation thresholds. We have also applied the "most labile protomer" rule to reinterpret-exclusively via classical charge-induced dissociation cleavages-several dissociation processes proposed for protonated molecules. In an accompanying letter, we have also applied a similar "most labile electromer" rule to ionized molecules.
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Steroids that take part in the pathways of human steroidogenesis are involved in many biological mechanisms where they interact with calcium. In the present work, the binding selectivities and affinities for calcium of progestagens, mineralocorticoids, androstagens, and estrogens were studied by Electrospray Ionization-Mass Spectrometry (ESI-MS). The adduct profile of each steroid was characterized by high resolution and tandem mass spectrometry. The relative stability of the most important adducts was studied by threshold collision induced dissociation, E1/2. Doubly-charged steroid-calcium complexes [nM + Ca]2+ with n = 1-6 were predominant in the mass spectra. The adduct [5M + Ca]2+ was the base peak for most 3-keto-steroids, while ligands bearing hindered ketones or α-hydroxy-ketones also yielded [nM + Ca + mH2O]2+ with n = 3-4 and m = 0-1. Principal component analysis allowed us to spot the main differences and similarities in the binding behavior of these steroids. The isomers testosterone and dehydroepiandrosterone, androstanolone and epiandrosterone, and 17-α-hydroxyprogesterone and 11-deoxycorticosterone showed remarkable differences in their adduct profiles. Computational modeling of representative adducts was performed by density functional theory methods. The possible binding modes at low and high numbers of steroid ligands were determined by calcium Gas Phase Affinity, and through modeling of the complexes and comparison of their relative stabilities, in agreement with the experimental results.
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Cálcio , Espectrometria de Massas por Ionização por Electrospray , Humanos , Cálcio/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Esteroides , CetonasRESUMO
The imaging of biological tissues can offer valuable information about the sample composition, which improves the understanding of analyte distribution in such complex samples. Different approaches using mass spectrometry imaging (MSI), also known as imaging mass spectrometry (IMS), enabled the visualization of the distribution of numerous metabolites, drugs, lipids, and glycans in biological samples. The high sensitivity and multiple analyte evaluation/visualization in a single sample provided by MSI methods lead to various advantages and overcome drawbacks of classical microscopy techniques. In this context, the application of MSI methods, such as desorption electrospray ionization-MSI (DESI-MSI) and matrix-assisted laser desorption/ionization-MSI (MALDI-MSI), has significantly contributed to this field. This review discusses the evaluation of exogenous and endogenous molecules in biological samples using DESI and MALDI imaging. It offers rare technical insights not commonly found in the literature (scanning speed and geometric parameters), making it a comprehensive guide for applying these techniques step-by-step. Furthermore, we provide an in-depth discussion of recent research findings on using these methods to study biological tissues.
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Microscopia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , LasersRESUMO
BACKGROUND AND AIM: Echinodorus macrophyllus (Kunth.) Micheli is popularly used for acute and chronic inflammatory conditions. The anti-inflammatory activity was previously demonstrated for its flavonoid-enriched fractions. The aim of this work assessed the antinociceptive properties of both aqueous extract and its fractions. EXPERIMENTAL PROCEDURE: The antinociceptive activity was determined by acetic acid-induced writhing, formalin test, tail immersion test, hot-plate test, xylene-induced ear edema methods, and the evaluation of its mechanism was performed in the writhing model. The aqueous extract of Echinodorus macrophyllus (AEEm) was fractionated, yielding Fr20, and Fr40. Fr40 composition was determined by HPLC-DAD-ESI-MS. RESULTS AND CONCLUSION: Fr20 (all doses) and Fr40 (100 mg/kg) reduced the nociception in the tail-flick model. Both fractions increased the percentage of maximum possible effect with 25 mg/kg, in the hot-plate assay, at 60 min, while AEEm reduced pain only with 50 and 100 mg/kg. There was a reduction in xylene-edema index, with Fr40 (25 mg/kg), AEEm (50 mg/kg) and Fr20 (50 mg/kg). All doses of AEEm, Fr20, and Fr40 reduced both phases of the formalin model. In the abdominal contortion model, Fr40 presented the highest activity, reducing 96% of contortions and its antinociceptive mechanism was evaluated. The results indicated the involvement of NO and adrenergic activation pathways. The main components of Fr40 are swertisin, swertiajaponin, isoorientin 7,3'-dimethyl ether, swertisin-O-rhamnoside, isoorientin, isovitexin, isovitexin-Orhamnoside, and isovitexin-7-O-glucoside. The aqueous extract of E. macrophyllus leaves and its fractions exhibited significant analgesic effect, mediated through both peripheral and central mechanisms being considered a potentially antinociceptive drug.
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A 25 µm i.d x 1.2 m length PS-DVB porous layer open tubular column (PLOT) was prepared and assessed in the configuration of a nano liquid chromatography coupled to an electron ionization mass spectrometry system (OT-nanoLC-EI-Ms), via the direct insertion of the column outlet into the ionization source. The developed system's operational parameters were comprehensively studied, and the setup performance was investigated employing both unidimensional and column switching configurations. As a result, the OT-nanoLC-EI-MS system demonstrated competitive applicability in separating non-amenable ESI compounds, such as polyaromatic hydrocarbons (PAHs) and non-amenable GC compounds such as thermolabile pesticides. Furthermore, with excellent chromatographic performance, the PLOT columns can work under more compatible EI-detection conditions - such as the elution with 100% organic solvent. For example, PAHs retention factors ranged between 1.5 and 2.2 for 100% MeCN mobile phase, and more than 33,000 plates per meter for naphthalene at 50 nL/min flow rate. In analyzing thermolabile pesticides, the column switching PLOT-nanoLC-EI-MS system provided LODs of 25 µg/L, demonstrating suitable intra e interday reproducibility (% RSD < 13%, n = 3), and possibilities the direct injection of raw samples with suitable robustness.
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Praguicidas , Espectrometria de Massas por Ionização por Electrospray , Elétrons , Porosidade , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
The biological and pharmacological properties of natural polyphenols of the extract of Euterpe oleracea stone (EEOS) are associated with the central nervous system (CNS). To investigate the sedative and myorelaxant activity of EEOS in vivo, this study aimed to present the myorelaxant and sedative effects of EEOS in Wistar rats using spontaneous locomotor activity and motor electrophysiology. A total of 108 animals were used in the following experiments: a) behavioral tests (n = 27); b) electromyographic recordings of skeletal muscle (n = 27); c) respiratory muscle activity recordings (n = 27); d) cardiac muscle activity recordings (n = 27). The behavioral characteristics were measured according to the latency time of onset, the transient loss of posture reflex and maximum muscle relaxation. Electrodes were implanted in the gastrocnemius muscle and in the tenth intercostal space for electromyographic (EMG) signal capture to record muscle contraction, and in the D2 lead for electrocardiogram acquisition. After using the 300 mg/kg dose of EEOS intraperitoneally, a myorelaxant activity exhibited a lower frequency of contractility with an amplitude pattern of low and short duration at gastrocnemius muscle and intercostal muscle, which clearly describes a myorelaxant activity and changes in cardiac activity. The present report is so far the first study to demonstrate the myorelaxant activity of this extract, indicating an alternative route for açai stone valorization and its application in pharmaceutical fields.
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Open tubular liquid chromatography (OT-LC) can provide superior chromatographic performance and more favorable mass spectrometry (MS) detection conditions. These features could provide enhanced sensitivity when coupled with electrospray ionization sources (ESI-) and lead to unprecedented detection capabilities if interfaced with a highly structural informative electron ionization (EI) source. In the past, the exploitation of OT columns in liquid chromatography evolved slowly. However, the recent instrumental developments in capillary/nanoLC-MS created new opportunities in developing and applying OT-LC-MS. Currently, the analytical advantages of OT-LC-MS are mainly exploited in the fields of proteomics and biosciences analysis. Nevertheless, under the right conditions, OT-LC-MS can also offer superior chromatographic performance and enhanced sensitivity in analyzing small molecules. This review will provide an overview of the latest developments in OT-LC-MS, focusing on the wide variety of employed separation mechanisms, innovative stationary phases, emerging column fabrication technologies, and new OT formats. In the same way, the OT-LC's opportunities and shortcomings coupled to both ESI and EI will be discussed, highlighting the complementary character of those two ionization modes to expand the LC's detection boundaries in the performance of targeted and untargeted studies.
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Cromatografia Líquida/métodos , Espectrometria de Massas , Escherichia coli/metabolismo , Proteômica/métodos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
An accurate and sensitive ultrasound-dispersive liquid-liquid microextraction technique followed by high-performance liquid chromatography separation coupled with electrospray ionization tandem mass spectrometry detection method to determine the presence of tetrabromobisphenol A (TBBPA) in complex environmental matrices is proposed. The miniaturized procedure was used to extract and quantify the analyte in domestic sewage, anaerobic sludge, and the aquatic test organism species Daphnia magna and Chironomus sancticaroli, which are standardized organisms for ecotoxicity bioassays. Limits of detection of 2 ng L-1 (domestic sewage), 2 ng g-1 (anaerobic sludge), 0.25 ng g-1 (D. magna), and 5 ng g-1 (C. tentans) were obtained. The presence of TBBPA was determined in domestic sewage and anaerobic sludge from an anaerobic batch bioreactor at a concentration of 0.2 ± 0.03 µg L-1 and 507 ± 79 ng g-1 , respectively. In D. magna and C. sancticaroli exposed to TBBPA in an acute toxicity bioassay, the micropollutant accumulated at 3.74 and 8.87 µg g-1 , respectively. The proposed method is a simple and cost-effective tool to determine TBBPA environmental occurrence and biomagnification potential compared with conventional extraction methods. To the best of our knowledge, this is the first liquid-liquid miniaturized extraction method to be applied to D. magna and C. sancticaroli. Environ Toxicol Chem 2020;39:2147-2157. © 2020 SETAC.
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Microextração em Fase Líquida/métodos , Bifenil Polibromatos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Calibragem , Chironomidae/metabolismo , Cromatografia Líquida de Alta Pressão , Daphnia/metabolismo , Limite de Detecção , Modelos Lineares , Padrões de Referência , Solventes/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Plasma and tissue from breast cancer patients are valuable for diagnostic/prognostic purposes and are accessible by multiple mass spectrometry (MS) tools. Liquid chromatography-mass spectrometry (LC-MS) and ambient mass spectrometry imaging (MSI) were shown to be robust and reproducible technologies for breast cancer diagnosis. Here, we investigated whether there is a correspondence between lipid cancer features observed by desorption electrospray ionization (DESI)-MSI in tissue and those detected by LC-MS in plasma samples. The study included 28 tissues and 20 plasma samples from 24 women with ductal breast carcinomas of both special and no special type (NST) along with 22 plasma samples from healthy women. The comparison of plasma and tissue lipid signatures revealed that each one of the studied matrices (i.e., blood or tumor) has its own specific molecular signature and the full interposition of their discriminant ions is not possible. This comparison also revealed that the molecular indicators of tissue injury, characteristic of the breast cancer tissue profile obtained by DESI-MSI, do not persist as cancer discriminators in peripheral blood even though some of them could be found in plasma samples.
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Neoplasias da Mama/metabolismo , Carcinoma Ductal/metabolismo , Metabolismo dos Lipídeos , Lipidômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/sangue , Carcinoma Ductal/sangue , Feminino , Humanos , Lipídeos/sangue , Pessoa de Meia-IdadeRESUMO
Documents with handwritten portions are often susceptible to adulteration, forgery, and addition of entries, raising a problem of social concern. In this study, DESI ionization with imaging capabilities is applied to identify fraud in handwritten documents made using erasable pens of the chemical method of erasing (other than the usual physical methods). A fraud procedure was simulated in which an original entry made in white office paper was erased and replaced with a new one. The areas were directly analyzed using a DESI-MSI ion source coupled to a Q-Extractive mass spectrometer. Chemical images were obtained mapping the intensity of selected ions, spelling out each part of the fraud process as irrefutable evidence of its occurrence. Thus, the potential application of DESI-MSI in detecting fraud in suspect documents is demonstrated as a useful, simple, and fast alternative for the traditional techniques employed in these situations.
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The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100â¯nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881⯱â¯3.7) and Control (883⯱â¯5.2) groups (pâ¯>â¯0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (pâ¯<â¯0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (pâ¯<â¯0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos.
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Bovinos/embriologia , Meios de Cultura/química , Técnicas de Cultura Embrionária/veterinária , Peptídeo Natriurético Tipo C/farmacologia , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Bovinos/genética , Feminino , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/química , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In this study, a molecularly imprinted polymer-coated probe electrospray ionization mass spectrometry (MIPCPESI-MS) method was developed for detection of phorbol esters (PEs) and deoxyphorbol metabolites in Jatropha curcas leaves. Such an approach was established by sticking on a metallic needle a molecularly imprinted polymer to particularly design a MIP-coated probe for selective sampling and ionization of PEs and deoxyphorbol metabolites. By a subsequent application of a high voltage and methanol, as spray solvent, ESI was generated for direct and rapid analysis under ambient and open-air conditions. MIP-coated probe exhibited a high sampling capacity of the PEs and its metabolites in methanolic extracts of J. curcas leaves compared with the non-imprinted polymer (NIP)-coated probe. MIPCPESI-MS allowed the detection of phorbol 12,13-diacetate (PDA) from J. curcas leaves with minimal sample preparation, and with detection limit and quantification reaching 0.28 µg/mL and 0.92 µg/mL, respectively. Also, good linearity was obtained with R2 > 0.99 and precision and accuracy values between 4.06-13.49% and - 1.60 to - 15.26%, respectively. The current method was successfully applied to screening methanolic extracts of six different J. curcas leaf genotypes (three toxic and three non-toxic). PDA and three PE deoxyphorbol metabolites were identified only from toxic genotypes, in which PDA was determined with concentration ranging from 222.19 ± 23.55 to 528.23 ± 19.72 µg/g. All these findings support that the MIPCPESI-MS method developed here has a high potential for the analysis of PEs in plant extracts enabling differentiation of toxic and non-toxic genotypes earlier in the leaves.
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Jatropha/química , Impressão Molecular/métodos , Ésteres de Forbol/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Limite de Detecção , Modelos Lineares , Compostos Fitoquímicos/química , Polímeros , Reprodutibilidade dos TestesRESUMO
Coconut oil (CO) from fifteen different varieties of coconuts (Cocos nucifera L.) and one CO processed on an industrial scale were analyzed by electrospray ionization mass spectrometry (ESI-MS) and the data processed using the chemometric tools principal component analysis and independent component analysis. ESI-MS fingerprinting of lipid compounds showed predominance of diacylglycerols and triacylglycerols, as confirmed by high-resolution MS measurements. Chemometric processing of the ESI-MS data differentiated the coconut oil samples, showing that different coconut varieties/cultivars produce oils with distinguishable abundances of lipidic compounds. Thus ESI-MS analysis followed by data treatment using chemometric tools offers a tool able to classify the industrial coconut oils in a fast, simple and effective way, as well as serving as a potential method to identify the coconut varieties by the CO origin, and the occurrence of any adulteration. The procedure may also be applied for quality control of the industrial processes.
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Cocos/química , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray , Óleo de Coco , Diglicerídeos/análise , Análise de Alimentos , Triglicerídeos/análiseRESUMO
The analyses of drugs and metabolites in complex matrices have been widely studied in recent years. However, due to high levels endogenous compounds and matrix complexity, these analyses require a sample pre-treatment step. To this aim, two lab-made extractive phases were integrated to probe electrospray ionization mass spectrometry (PESI-MS) technique for direct analysis of illicit drugs in biological fluids and phorbol esters in Jatropha curcas extract. The polypyrrole (PPy) phase was electropolymerized onto a platinum wire surface by cyclic voltammetry. The molecularly imprinted polymer (MIP) was synthesized and adhered onto a stainless-steel needle with epoxy resin. The PPy-PESI-MS method showed to be linear in a concentration range from 1 to 500⯵g L-1, with accuracy values between -2.1 and 14%, and precision values between 0.8 and 10.8%. The MIP-PESI-MS method showed to be linear in a concentration range from 0.9 to 30â¯mgâ¯L-1, with accuracy values between -1.6 and -15.3%, and precision values between 4.1 and 13.5%.
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Impressão Molecular/métodos , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/isolamento & purificação , Polímeros/química , Pirróis/química , Microextração em Fase Sólida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Cocaína/análise , Cocaína/isolamento & purificação , Voluntários Saudáveis , Humanos , Jatropha/química , Dietilamida do Ácido Lisérgico/análise , Dietilamida do Ácido Lisérgico/isolamento & purificação , Metanfetamina/análise , Metanfetamina/isolamento & purificação , N-Metil-3,4-Metilenodioxianfetamina/análise , N-Metil-3,4-Metilenodioxianfetamina/isolamento & purificação , Ésteres de Forbol/análise , Ésteres de Forbol/isolamento & purificação , Extratos Vegetais/análise , Extratos Vegetais/isolamento & purificação , Saliva/metabolismo , Aço Inoxidável/química , UrináliseRESUMO
Inorganic polymers in aqueous solutions are being proposed as essential components in new theories concerning nonclassical nucleation and growth of nanominerals relevant to environmental nanogeosciences. The study of those complex natural processes requires multi-technique analytical approaches able to characterize the solutions and their constituents (solutes, oligomers, polymers, clusters and nanominerals) from atomic to micrometric scales. A novel analytical approach involving an electrospray ionization source (ESI) coupled to time-of-flight mass spectrometry (TOF/MS) was developed to identify inorganic polymers in aqueous solution. To this end, the presence of initial Al oligomers and their polymerization processes was studied during a nanomineral aqueous synthesis (hydrobasaluminte, Al4 SO4 (OH)10 ·12-36H2 O). Ensuring the feasibility and robustness of the methodology as well as the stability of the polymers under study (avoiding undesirable fragmentation), a meticulous study of the ESI-TOF MS working conditions was performed. Precision of the methodology was evaluated obtaining relative standard deviations below 3.3%. For the first time in the study of inorganic polymers in the earth sciences, the mass accuracy error (ppm) has been reported and the use of significant decimal figures of the m/z signal has been taken into account. Complementary to this, a four-step polymer assignment methodology and a database with the Al- and Al-SO4 2- polymers assigned were created. Several polymers have been assigned for the first time, including Al (SO4 )+ ·H2 O, Al2 O(SO4 )2+ ·H2 O, Al5 O4 (OH)5 2+ ·2H2 O, and Al3 O5 (OH)2- ·4H2 O, among others. The results obtained in the present study help create a foundation to include mass spectrometry as a routine analytical technique to study mineral formation in aqueous solution.
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A method combining liquid chromatography with a dual-probe ultraspray electrospray ionization (ESI) source and time-of-flight high-resolution mass spectrometry (LC-ESI-TOF/MS) was developed for the simultaneous determination of four steroidal sex hormones, estrone (E1), 17ß-estradiol (E2), 17α-ethinyl estradiol (EE2), and estriol (E3), as well as five of their hydroxylated metabolites, 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 16α-hydroxyestrone (16-OHE1), 2-hydroxyestradiol (2-OHE2), and 4-hydroxyestradiol (4-OHE2), in water samples in a short chromatographic run of 10 min. Derivatization of the analytes was optimized using dansyl chloride as the derivatizing agent. Under optimal positive ionization conditions, the following signals, which had not been previously reported, were observed (with theoretical values of m/z 377.1373 for 2- and 4-OHE1 and 378.1452 for 2- and 4-OHE2), corresponding to doubly derivatized catechol estrogens in the form of [M+2H]2+. These mass spectrometric signals were more abundant than those reported previously for the [M+H]+ forms of these hydroxylated metabolites. Solid-phase extraction (SPE) with an octadecyl-endcapped sorbent was used to pretreat tap water and effluent from a wastewater treatment plant (WWTP) in Santiago, Chile. The method achieved the simple, fast, and sensitive measurement of nine estrogens with quantitative recoveries (higher than 85.4%). Detection and quantification limits were between 1 and 17 ng L-1 and between 3 and 58 ng L-1, respectively, for all compounds in water. The estrogens E1 and E2 were found in WWTP effluent at concentrations of 7 ± 1 and 41 ± 1 ng L-1, respectively, and EE2 was detected at a concentration below the limit of quantitation. This study shows that the proposed method is suitable for the accurate, rapid, and selective determination of all these analytes at trace levels. Graphical abstract á .
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Compostos de Dansil/química , Estrogênios/análise , Estrogênios/classificação , Águas Residuárias/análise , Água/análise , Chile , Cromatografia Líquida/métodos , Hidroxilação , Limite de Detecção , Espectrometria de Massas em Tandem/métodos , Fatores de TempoRESUMO
The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV-Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI-MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery. Graphical Abstract á .
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Metabolismo Secundário , Espectrometria de Massas por Ionização por Electrospray/métodos , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Bacillus/efeitos dos fármacos , Humanos , Metabolômica/métodos , Micoses/tratamento farmacológico , Penicillium/efeitos dos fármacos , Polienos/química , Polienos/metabolismo , Polienos/farmacologia , Streptomyces/química , Espectrometria de Massas em Tandem/métodos , Xantenos/química , Xantenos/metabolismo , Xantenos/farmacologiaRESUMO
Background: A new ι-carrageenase-producing strain was screened from mangroves and authenticated as Pseudoalteromonas carrageenovora ASY5 in our laboratory. The potential application of this new strain was evaluated. Results: Medium compositions and culturing conditions in shaking flask fermentation were firstly optimized by single-factor experiment. ι-Carrageenase activity increased from 0.34 U/mL to 1.08 U/mL after test optimization. Optimal fermentation conditions were 20°C, pH 7.0, incubation time of 40 h, 15 g/L NaCl, 1.5% (w/v) yeast extract as nitrogen source, and 0.9% (w/v) ι-carrageenan as carbon source. Then, the crude ι-carrageenase was characterized. The optimum temperature and pH of the ι-carrageenase were 40°C and 8.0, respectively. The enzymatic activity at 3540°C for 45 min retained more than 40% of the maximum activity. Meanwhile, The ι-carrageenase was inhibited by the addition of 1 mmol/L Cd2+ and Fe3+ but increased by the addition of 1 mmol/L Ag+, Ba2+, Ca2+, Co2+, Mn2+, Zn2+, Fe2+, and Al3+. The structure of oligosaccharides derived from ι-carrageenan was detected using electrospray ionization mass spectrometry (ESI-MS). The ι-carrageenase degraded ι-carrageenan, yielding disaccharides and tetrasaccharides as main products. Conclusions: The discovery and study of new ι-carrageenases are beneficial not only for the production of ι-carrageenan oligosaccharides but also for the further utilization in industrial production.
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Proteínas de Bactérias/metabolismo , Pseudoalteromonas/enzimologia , Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/biossíntese , Temperatura , Carbono/metabolismo , Carragenina/biossíntese , Espectrometria de Massas por Ionização por Electrospray , Fermentação , Concentração de Íons de Hidrogênio , Hidrólise , Nitrogênio/metabolismoRESUMO
Amino acids play an important role in clinical analysis. Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) has proven to possess several characteristics that make it a powerful and useful tool for the analysis of amino acids in clinical studies. Here we present a method for the separation and quantitative analysis of 27 amino acids in urine based on CE-ESI-MS. The method presents an improved resolution between the isomers Leu, Ile, and aIle, in comparison to other CE-ESI-MS methods in the literature. This method is fast, selective, and simple and has improved sensitivity by applying a pH-mediated stacking strategy, showing that it can be successfully used for amino acid analysis and probably for other small cationic metabolites.
Assuntos
Aminoácidos/urina , Eletroforese Capilar/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Humanos , Concentração de Íons de Hidrogênio , Isoleucina/urina , Leucina/urinaRESUMO
The bacteria of the genus Curtobacterium are usually seen as plant pathogen, but some species have been identified as endophytes of different crops and could as such present a potential for disease control and plant growth promotion. We have therefore applied the desorption electrospray ionization mass spectrometry imaging (DESI-MSI) in the direct analysis of living Curtobacterium sp. strain ER1/6 colonies to map the surface metabolites, and electrospray ionization tandem mass spectrometry (ESI-MS/MS) for characterization of these compounds. Several colony-associated metabolites were detected. The ESI-MS/MS showed characteristic fragmentations for phospholipids including the classes of glycerophosphocholine, glycerophosphoglycerol, and glycerophosphoinositol as well as several fatty acids. Although a secure identification was not obtained, many other metabolites were also detected for this bacteria species. Principal component analysis showed that fatty acids were discriminatory for Curtobacterium sp. ER1/6 during inoculation on periwinkle wilt (PW) medium, whereas phospholipids characterize the bacterium when grown on the tryptic soy agar (TSA) medium.