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Carcinoid syndrome represents the most common functional syndrome that affects patients with neuroendocrine neoplasms. Its clinical presentation is really heterogeneous, ranging from mild and often misdiagnosed symptoms to severe manifestations, that significantly worsen the patient's quality of life, such as difficult-to-control diarrhoea and fibrotic complications. Serotonin pathway alteration plays a central role in the pathophysiology of carcinoid syndrome, accounting for most clinical manifestations and providing diagnostic tools. Serotonin pathway is complex, resulting in production of biologically active molecules such as serotonin and melatonin, as well as of different intermediate molecules and final metabolites. These activities require site- and tissue-specific catalytic enzymes. Variable expression and activities of these enzymes result in different clinical pictures, according to primary site of origin of the tumour. At the same time, the biochemical diagnosis of carcinoid syndrome could be difficult even in case of typical symptoms. Therefore, the accuracy of the diagnostic methods of assessment should be improved, also attenuating the impact of confounding factors and maybe considering new serotonin precursors or metabolites as diagnostic markers. Finally, the prognostic role of serotonin markers has been only evaluated for its metabolite 5-hydroxyindole acetic acid but, due to heterogeneous and biased study designs, no definitive conclusions have been achieved. The most recent progress is represented by the new therapeutic agent telotristat, an inhibitor of the enzyme tryptophan hydroxylase, which blocks the conversion of tryptophan in 5-hydroxy-tryptophan. The present review investigates the clinical significance of serotonin pathway in carcinoid syndrome, considering its role in the pathogenesis, diagnosis, prognosis and therapy.
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Síndrome do Carcinoide Maligno/metabolismo , Fenilalanina/análogos & derivados , Pirimidinas/uso terapêutico , Serotonina/metabolismo , Transdução de Sinais , Triptofano Hidroxilase/antagonistas & inibidores , Humanos , Síndrome do Carcinoide Maligno/diagnóstico , Síndrome do Carcinoide Maligno/tratamento farmacológico , Síndrome do Carcinoide Maligno/fisiopatologia , Fenilalanina/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
Proteomics-based mass spectrometry has gained increasing amounts of popularity in recent years. In particular, high resolution accurate mass measurements in mass spectrometry has gained notoriety for giving the capability of high throughput analysis with lower cost to the user. In particular, its uses in the identification of protein sequence through the utilization of bottom-up, middle-down, and top-down approaches has been widely discussed. In this chapter, we discuss the advantages of each technique as well as using the techniques in tandem to gain well-rounded structural data on our protein of interest, glucokinase. The study will focus on the use of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry, but give insights into the advantages that may come from the utilization of other high resolution techniques.
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Glucoquinase/química , Espectrometria de Massas , Análise de Sequência de Proteína , ProteômicaRESUMO
We report a sensory platform for the determination of common explosive species (e.g., TNT, PETN, RDX) based on the differential response from two different luminescent metal nanoclusters. In particular, whereas the red emission from bovine serum albumin-protected gold nanoclusters was strongly quenched by nitro-, nitrate-, and nitroamine-containing explosive organic molecules, blue-emitting glutathione-capped copper nanoclusters proved inert to quenching by these same analytes, instead showing evidence for aggregation-induced emission enhancement (AIEE). As a result, this discrete gold/copper nanocluster pairing provides a dual-probe, ratiometric (red-to-blue) system signaling the presence of TNT and other common explosives. This strategy opens up new potential for nanocluster-based analyte signaling, with implications to fluorescence resonance energy transfer (FRET) strategies as well.
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Stercobilin is an end-stage metabolite of hemoglobin, a component of red blood cells. It has been found that there is a significantly lower concentration of stercobilin in the urine of people diagnosed with autism spectrum disorders, suggesting potential use as a biomarker. In vitro, we have synthesized stercobilin from its precursor bilirubin through a reduction reaction proceeded by an oxidation reaction. In addition, we have isotopically labeled the stercobilin product with deuterium using this protocol. Nuclear magnetic resonance investigations show the products of the unlabeled stercobilin (Rxn 1) and the deuterated stercobilin (Rxn 2) both had a loss of signals in the 5.0- to 7.0-ppm range indicating proper conversion to stercobilin. Changes in the multiplicity of the sp3 region of the proton nuclear magnetic resonance suggest proper deuterium incorporation. Mass spectrometry studies of Rxn 1 show a difference in fragmentation patterns than that of Rxn 2 proposing potential locations for deuterium incorporation. This isotopologue of stercobilin is stable (>6 mo), and further analysis permits investigation for its use as a biomarker and potential quantitative diagnostic probe for autism spectrum disorders.
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As a relatively recent research field, plant metabolomics has gained increasing interest in the past few years and has been applied to answer biological questions through large-scale qualitative and quantitative analyses of the plant metabolome. The combination of sensitivity and selectivity offered by mass spectrometry (MS) for measurement of many metabolites in a single shot makes it an indispensable platform in metabolomics. In this regard, Fourier-transform ion cyclotron resonance (FTICR) has the unique advantage of delivering high mass resolving power and mass accuracy simultaneously, making it ideal for the study of complex mixtures such as plant extracts. Here we optimize soybean leaf extraction methods compatible with high-throughput reproducible MS-based metabolomics. In addition, matrix-assisted laser desorption ionization (MALDI) and direct LDI of soybean leaves are compared for metabolite profiling. The extraction method combined with electrospray (ESI)-FTICR is supported by the significant reduction of chlorophyll and its related metabolites as the growing season moves from midsummer to the autumn harvest day. To our knowledge for the first time, the use of ESI-FTICR MS and MALDI-FTICR MS is described in a complementary manner with the aim of metabolic profiling of plant leaves that have been collected at different time points during the growing season.
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Análise de Fourier , Glycine max/metabolismo , Ensaios de Triagem em Larga Escala , Metabolômica/métodos , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Ciclotrons , Extratos Vegetais/química , Folhas de Planta/química , Glycine max/químicaRESUMO
RATIONALE: Bilin tetrapyrroles including stercobilin are unique to mammalian waste; they have been used as markers of source water contamination and may have important diagnostic value in human health conditions. Unfortunately, commercial isotopomers for bilins are not available. Thus, there is a need for isotopomer standards of stercobilin and other bilins for quantification in environmental and clinical diagnostic applications. METHODS: A procedure is described here using H2 (18) O to label the carboxylic acid groups of bilin tetrapyrroles. Reaction conditions as a function of temperature and reagent volume were found to produce a mixture of isotopomers, as assessed by electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Stability as a function of storage time and temperature and in conjunction with solid-phase extraction (SPE) was assessed. RESULTS: The highest labeling efficiency was achieved at 70 °C for 8 h, while a stable ratio of the isotopmers could be produced at 60 °C for 4 h. The stability of the isotopic distribution was maintained under storage (room temperature or frozen) for 20 days. It was also stable throughout SPE. The high mass accuracy and resolving power of FTICRMS enables clear distinction between (18) O-labeled bilins from other unlabeled bilins present, avoiding a potential interference in quantitation. CONCLUSIONS: A procedure was developed to label bilins with (18) O. The final ratio of the (18) O-labeled bilin isotopomers was reproducible and highly stable for at least 20 days under storage. This ratio was not changed in any statistically significant way even after SPE. Thus a reliable method for producing stable isotopomer ratios for bilins has been achieved. Copyright © 2016 John Wiley & Sons, Ltd.
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Pigmentos Biliares/química , Metabolômica , Espectrometria de Massas por Ionização por Electrospray , Animais , Humanos , TetrapirróisAssuntos
Microbioma Gastrointestinal , Glutens , Encéfalo/metabolismo , Disbiose , Glutens/metabolismo , Humanos , PeptídeosRESUMO
Placentophagia, ingestion of placenta and amniotic fluid, usually during parturition, is a behavioral feature of nearly all nonaquatic, placental mammals, and is a nexus for several interlocking behavioral phenomena. Placentophagia has not been typical of human cultures, but in recent years, some women in affluent societies have engaged in it, thereby bringing publicity to the behavior. First, we summarized benefits of placentophagia for nonhuman mammals, which include increased attractiveness of neonates, enhanced onset of maternal behavior, suppression of pseudopregnancy, and enhancement of opioid hypoalgesia by Placental Opioid-Enhancing Factor (POEF), a benefit that may extend well outside the context of parturition. The research on POEF in animals was discussed in detail. Then we discussed placentophagia (placentophagy) in humans, and whether there is validity to the claims of various benefits reported primarily in the pro-placentophagy literature, and, although human afterbirth shows POEF activity, the POEF effect has not yet been tested in humans. Finally, we discussed the general possible implications, for the management of pain and addiction, of isolating and characterizing POEF.
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Analgésicos Opioides , Placenta , Animais , Recém-Nascido , Feminino , Gravidez , Humanos , Período Pós-Parto , Dor , Comportamento Materno , MamíferosRESUMO
RATIONALE: Bilins are metabolic products of hosts and bacteria on porphyrins, and are markers of health state and human waste contamination. Although bilin tandem mass spectrometry reports exist, their fragmentation behavior as a function of structure has not been compared, nor has fragmentation been examined as a function of collision energy. METHODS: The fragmentation of bilins generated by positive ion mode electrospray ionization is examined by collision-induced dissociation (CID). CID on a quadrupole ion trap and on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer as a function of collision energy is compared. Methyl esterification was used to deduce which product ions contain the inner pyrrole rings. FT-ICR high mass accuracy measurements were used to determine the formulas of the resultant product ions. RESULTS: The central carbon's bonding to the inner pyrrole rings influences fragmentation. Bilirubin is unique because fragmentation adjacent to the central methylene group between innermost rings predominates, and loss of a terminal pyrrole is observed only with helium collision gas. The other bilins lose the terminal pyrroles first; as CID energy is increased, additional fragmentation due to neutral losses of small molecules such as H(2)O, CO, CO(2), and methanol occurs. CONCLUSIONS: Based on these observations, fragmentation schemes for the bilins are proposed that are strongly dependent on the molecular structure and collision energy; only bilirubin fragmentation is influenced significantly by the collision gas used. This report should have value in identification of this class of molecules for biomarker detection.
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Pigmentos Biliares/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Ésteres/química , Humanos , Íons/química , Modelos MolecularesRESUMO
Drought is the most prolific form of abiotic stress that legumes and cereal plants alike can endure, and the planting of an improper cultivar at the beginning of a season can cause unexpected losses up to fifty percent under water deficient conditions. Herein, a plant introduction (PI) of an exotic cultivar of soybean (Glycine max), PI 567731, which demonstrates a slow wilting (SW) canopy phenotype in maturity group III, was profiled under drought conditions in field trials in Missouri against a drought susceptible check cultivar, Pana. Metabolomic profiling was carried out on samples of leaves from each of these cultivars at V5 and R2 growth stages both while irrigated and while under drought stress for three weeks. PI 567731 was observed to have differential phytochemical content, and enhanced levels of chlorophyll (Chl) a/b and pheophytin (Pheo) were profiled by direct infusion electrospray Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Indicating drought induced changes of the photosystem and photosynthetic capabilities alongside water preservation strategies are important within the SW phenotype drought response. Subsequent multivariate analysis was able to form predictive models, encompassing the variance of growth and drought stress of the cultivar. Moreover, the existence of unique Chl-related metabolites (CRM) (m/z > 900) were confirmed through tandem mass spectrometry. The resultant coordination of fatty acids to the core of the porphyrin ring was observed and played an unknown role in the proliferation of the photosynthesis. However, the relative ratio of the most abundant CRM is undisturbed by drought stress in PI 567731, in contrast to the drought susceptible cultivar. These results provide key insights into drought related metabolic mechanisms.
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The study of biological specimens by mass spectrometry imaging (MSI) has had a profound influence in the various forms of spatial-omics over the past two decades including applications for the identification of clinical biomarker analysis; the metabolic fingerprinting of disease states; treatment with therapeutics; and the profiling of lipids, peptides and proteins. No singular approach is able to globally map all biomolecular classes simultaneously. This led to the development of many complementary multimodal imaging approaches to solve analytical problems: fusing multiple ionization techniques, imaging microscopy or spectroscopy, or local extractions into robust multimodal imaging methods. However, each fusion typically requires the melding of analytical information from multiple commercial platforms, and the tandem utilization of multiple commercial or third-party software platforms-even in some cases requiring computer coding. Herein, we report the use of matrix-assisted laser desorption/ionization (MALDI) in tandem with desorption electrospray ionization (DESI) imaging in the positive ion mode on a singular commercial orthogonal dual-source Fourier transform ion cyclotron resonance (FT-ICR) instrument for the complementary detection of multiple analyte classes by MSI from tissue. The DESI source was 3D printed and the commercial Bruker Daltonics software suite was used to generate mass spectrometry images in tandem with the commercial MALDI source. This approach allows for the generation of multiple modes of mass spectrometry images without the need for third-party software and a customizable platform for ambient ionization imaging. Highlighted is the streamlined workflow needed to obtain phospholipid profiles, as well as increased depth of coverage of both annotated phospholipid, cardiolipin, and ganglioside species from rat brain with both high spatial and mass resolution.
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Many neuropeptides lack suitable amino acid residues for modification by existing selective isotope labeling methods and use in relative quantitation by mass spectrometry. To address this issue, a new stable isotope labeling method that targets tyrosine residues by coupling with light cysteine (d(0)) or heavy cysteine (d(2)) in the presence of tyrosinase was developed. Optimal derivatization conditions for 1microM leucine-enkephalin were achieved when 10mM cysteine and 200U/ml tyrosinase at pH 6.8 to 7.2 were used for a 60-min incubation period at room temperature. Under these conditions, leucine-enkephalin present at concentrations as low as 125nM was successfully labeled. When comparisons between the lightly labeled (d(0)) and heavily labeled (d(2)) forms were made, a discrepancy between the actual concentration ratio and the raw peak intensity ratio was observed; this is due to the overlap of an isotopic peak of the d(0) with the monoisotopic peak of d(2). Fortunately, this discrepancy can be corrected by one of two simple computational approaches described. The quantitative labeling of this method to neuropeptides with the terminal tyrosine was confirmed and provides an alternative when other selective isotope-coded affinity tagging methods are not suitable.
Assuntos
Cisteína/química , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Neuropeptídeos/análise , Tirosina/química , Sequência de Aminoácidos , Cromatografia Líquida , Encefalina Leucina/análise , Encefalina Leucina/química , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/metabolismo , Neuropeptídeos/químicaRESUMO
Hydroxylated polybrominated diphenyl ether (OH-PBDEs) metabolites have the potential to cause endocrine disruption as well as other health effects. Currently, gas chromatography/mass spectrometry (GC/MS) after derivatization is used for the analysis of OH-PBDEs. However, there is a need for the direct analysis of OH-PBDEs at relatively low concentrations in environmental and biological samples. Liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS) was evaluated for the analysis of nine OH-PBDEs, ranging from tri- to hexabrominated. Separation of the nine isomeric metabolites was achieved with reversed-phase liquid chromatography, followed by detection by APCI-MS in negative mode. Notably, a significant decrease in ionization was observed in 6-hydroxyl-substituted PBDE metabolites in the presence of an ortho-substituted bromine, relative to the other hydroxylated metabolites. This is probably due to the formation of dioxins in the source as a result of the high-temperature conditions, which prevented ionization by hydrogen abstraction. The MS/MS experiments also provided evidence of the neutral losses of HBr and Br(2), indicating the possible use of neutral loss scanning and selected reaction monitoring (SRM) for the screening of brominated metabolites in samples. The applicability of LC/APCI-MS/MS was demonstrated for the analysis of metabolites of BDEs 47 and 99 formed in human liver microsomes. The LC/APCI-MS/MS method was able to detect metabolites that had previously been identified by GC/MS following derivatization.
Assuntos
Cromatografia Líquida/métodos , Éteres Difenil Halogenados/química , Espectrometria de Massas em Tandem/métodos , Éteres Difenil Halogenados/metabolismo , Humanos , Hidroxilação , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura MolecularRESUMO
The field of ambient ionization mass spectrometry has witnessed the development of many novel and capable methods for the analysis and imaging of surfaces, with desorption electrospray ionization being a prominent technique that has been commercialized. The adaptation of this technique to existing mass spectrometry platforms requires a laboratory-built solution manufactured with the capability of fine, stable adjustments of the electrospray emitter for liquid or solid sampling purposes. The development, fabrication, and machining require tens of hours of labor for many custom solutions. Herein described is a highly modifiable alternative approach for the fabrication of a desorption electrospray ionization source, using computer-aided design and fused deposition modeling to three-dimensionally print a source platform that utilizes standard accessories of a commercial Bruker Daltonics mass spectrometer. Three-dimensional printing allows for the inexpensive, rapid development of highly modifiable plastic parts, with the total printing time of the apparatus requiring a singular day and only a few dollars of material using a consumer grade printer. To demonstrate the utility of this printed desorption electrospray ionization source, it was fitted on an unmodified Fourier transform ion cyclotron resonance mass spectrometer for a lipid fingerprint analysis in serial sections of rat brain tissue, with the acquisition of line scans of dye-coated slides for the demonstration of serial acquisition.
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Destruction of myelin, or demyelination, is a characteristic of traumatic spinal cord injury and pathognomonic for primary demyelinating pathologies such as multiple sclerosis (MS). The regenerative process known as remyelination, which can occur following demyelination, fails as MS progresses. Models of focal demyelination by local injection of gliotoxins have provided important biological insights into the demyelination/remyelination process. Here, injection of lysolecithin to induce spinal cord demyelination is investigated using matrix-assisted laser desorption/ionization mass spectrometry imaging. A segmentation analysis revealed changes to the lipid composition during lysolecithin-induced demyelination at the lesion site and subsequent remyelination over time. The results of this study can be utilized to identify potential myelin-repair mechanisms and in the design of therapeutic strategies to enhance myelin repair.
Assuntos
Doenças Desmielinizantes/patologia , Bainha de Mielina/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Medula Espinal/patologia , Animais , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Feminino , Lipídeos/análise , Lisofosfatidilcolinas/efeitos adversos , Camundongos Endogâmicos BALB C , Bainha de Mielina/química , Remielinização , Medula Espinal/químicaRESUMO
While the metabolism and excretion of polybrominated diphenyl ethers (PBDEs) have been reported in rodents, PBDE metabolism in humans has only recently been investigated. In this present study, individual human liver microsomes were incubated for 120 min with radiolabeled and nonradiolabeled BDE 47, 99, or 153 to determine their relative degrees of metabolism and to identify the structures of metabolites formed. Radiolabeled samples were analyzed using high-performance liquid chromatography/radiochemical detection, while nonradiolabeled samples were analyzed with and without derivatization using gas chromatography/mass spectrometry. Results from radiolabeled incubations demonstrated that human liver microsomes metabolized BDEs 47 and 99 but not BDE 153. Differences in the extent of BDE metabolism by the three individual liver specimens used in the study were observed. BDE 47 metabolized to a dihydroxylated BDE 47 and 2,4-dibromophenol, while BDE 99 metabolized to a dihydroxylated BDE 99, 2,4,5-tribromophenol and 1,3-dibromobenzene. This study showed that BDEs 47 and 99 are metabolized by human liver microsomes with relatively large interindividual differences. Results of this study could provide one explanation for the high bioaccumulation rate of BDE 153 in humans.
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Éteres Difenil Halogenados/metabolismo , Microssomos Hepáticos/metabolismo , Bifenil Polibromatos/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Éteres Difenil Halogenados/química , Humanos , Masculino , Pessoa de Meia-Idade , Bifenil Polibromatos/química , Adulto JovemRESUMO
Hafnium oxide (hafnia) exhibits unique enrichment properties towards phosphorylated peptides that are complementary to those of titanium oxide (titania) and zirconium oxide (zirconia) for use with mass spectrometric analysis in the field of proteomics.
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Caseínas/análise , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Bovinos , Háfnio , Humanos , Óxidos , Fosforilação , Titânio , ZircônioRESUMO
Enzyme linked immunosorbent assay (ELISA) was used for the analysis of tetracycline, chlortetracycline, oxytetracycline, and their transformed compounds in environmental water samples. The antibodies employed in ELISA showed high relative affinity for tetracycline, epitetracycline, chlortetracycline, and epichlortetracycline as compared to anhydrotetracycline, epianhydrotetracycline, and anhydrochlortetracycline. The specificity and crossreactivity of these antibodies are discussed in relation to the electrostatic potentials and average local ionization energies computed on the molecular surfaces of tetracycline antibiotics and their transformed compounds with an objective of identifying common features as well as differences that may be related to the experimentally observed variation in cross-reactivity values. The computations were performed at both the HF/STO-3G and HF/6-31+G* levels using the Gaussian 98 program. The results in this study are based upon molecular electrostatic potentials and local ionization energies computed on isodensity molecular surfaces. The surface electrostatic potentials are characterized in terms of a group of statistically defined quantities, which include the average deviation, the positive, negative, and total variances, positive and negative surface extrema, and a parameter indicating the degree of electrostatic balance.
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Antibacterianos/química , Anticorpos/química , Sítios de Ligação de Anticorpos , Modelos Moleculares , Eletricidade Estática , Tetraciclina/química , Antibacterianos/análise , Antibacterianos/imunologia , Anticorpos/imunologia , Simulação por Computador , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Conformação Molecular , Propriedades de Superfície , Tetraciclina/análise , Tetraciclina/imunologiaRESUMO
The formation of disulfide-bonds is vital for the proper folding of most secreted proteins and the stabilization of the final protein structure, including many of medical importance. The determination of disulfide-bonds is an important aspect of gaining a comprehensive understanding of the chemical structure of a protein. A long-term goal of ours is to examine the mechanism of disulfide-bond formation in aqueous solution and the potential role hydrogen bonds play in this process. Here, we report preliminary results from a method that utilizes the oxidizing power of iodine to generate disulfide bonds from synthesized model compounds, which is followed by nanoelectrospray ionization (nanoESI)- mass spectrometry (MS). By continuously monitoring the reaction mixture during disulfide formation, this nanoESI approach provides insight on the sequence of intermediate species formed, and how hydrogen-bonding donor/acceptor pairs may promote disulfide bond formation.
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Dissulfetos/química , Ligação de Hidrogênio , Microquímica/métodos , Modelos Químicos , Nanotecnologia/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Simulação por Computador , Projetos PilotoRESUMO
A sensitive, precise and accurate method for the quantification of the alimentary opioid peptide Gluten Exorphin B5 (GE-B5, Tyr-Gly-Gly-Trp-Leu) in cerebrospinal fluid (CSF) was developed using liquid chromatography-mass spectrometry (LC-MS). Aliquots (10 microL) of sheep CSF were injected into a LC-MS instrument equipped with a reversed-phase C12 column at a flow rate of 250 microL/min. The mobile phase consisted of Eluent A water with 0.01% acetic acid as an ion-pairing reagent, and Eluent B acetonitrile. The LC-MS system was programmed to divert column flow to waste for 3.5 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. DADLE (Tyr-D-Ala-Gly-Phe-D-Leu) was used as Internal Standard. No significant interfering peaks were detected at the retention times of GE-B5 in CSF blanks. The calibration curves were linear in the range of 0.39-78.00 ng/mL. The lower limit of detection and the lower limit of quantitation values for GE-B5 in CSF were established at 0.30 and 0.78 ng/mL, respectively. The intra-day and inter-day precision values were <12% relative standard deviation. The intra-day and inter-day accuracy were 99.46-100.86% and 98.95-100.02%, respectively. Recovery of GE-B5 in CSF samples was greater than 80%. Stability studies indicate that GE-B5 in CSF undergoes significant degradation (>55% after 600 min), which is reduced by the addition of protease inhibitors. This is the first reported method for the quantification of GE-B5 in CSF.