RESUMO
We present a mass spectrometry imaging (MSI) approach for the comprehensive mapping of neurotransmitter networks in specific brain regions. Our fluoromethylpyridinium-based reactive matrices facilitate the covalent charge-tagging of molecules containing phenolic hydroxyl and/or primary or secondary amine groups, including dopaminergic and serotonergic neurotransmitters and their associated metabolites. These matrices improved the matrix-assisted laser desorption/ionization (MALDI)-MSI detection limit toward low-abundance neurotransmitters and facilitated the simultaneous imaging of neurotransmitters in fine structures of the brain at a lateral resolution of 10 µm. We demonstrate strategies for the identification of unknown molecular species using the innate chemoselectivity of the reactive matrices and the unique isotopic pattern of a brominated reactive matrix. We illustrate the capabilities of the developed method on Parkinsonian brain samples from human post-mortem tissue and animal models. The direct imaging of neurotransmitter systems provides a method for exploring how various neurological diseases affect specific brain regions through neurotransmitter modulation.
Assuntos
Neurotransmissores/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Limite de Detecção , Doença de Parkinson/metabolismo , Primatas , RatosRESUMO
The progressive accumulation, aggregation, and spread of α-synuclein (αSN) are common hallmarks of Parkinson's disease (PD) pathology. Moreover, numerous proteins interact with αSN species, influencing its toxicity in the brain. In the present study, we extended analyses of αSN-interacting proteins to cerebrospinal fluid (CSF). Using coimmunoprecipitation, followed by mass spectrometry, we found that αSN colocalize with apolipoproteins on lipoprotein vesicles. We confirmed these interactions using several methods, including the enrichment of lipoproteins with a recombinant αSN, and the subsequent uptake of prepared vesicles by human dopaminergic neuronal-like cells. Further, we report an increased level of ApoE in CSF from early PD patients compared with matched controls in 3 independent cohorts. Moreover, in contrast to controls, we observed the presence of ApoE-positive neuromelanin-containing dopaminergic neurons in substantia nigra of PD patients. In conclusion, the cooccurrence of αSN on lipoprotein vesicles, and their uptake by dopaminergic neurons along with an increase of ApoE in early PD, proposes a mechanism(s) for αSN spreading in the extracellular milieu of PD.
Assuntos
Apolipoproteínas E/líquido cefalorraquidiano , Apolipoproteínas/líquido cefalorraquidiano , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/líquido cefalorraquidiano , Substância Negra/metabolismo , alfa-Sinucleína/líquido cefalorraquidiano , Idoso , Sequência de Aminoácidos , Apolipoproteínas/genética , Apolipoproteínas E/genética , Estudos de Casos e Controles , Estudos de Coortes , Neurônios Dopaminérgicos/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Melaninas/líquido cefalorraquidiano , Melaninas/genética , Pessoa de Meia-Idade , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ligação Proteica , Transporte Proteico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Substância Negra/patologia , Vesículas Transportadoras/metabolismo , alfa-Sinucleína/genéticaRESUMO
There is a high need to develop quantitative imaging methods capable of providing detailed brain localization information of several molecular species simultaneously. In addition, extensive information on the effect of the blood-brain barrier on the penetration, distribution and efficacy of neuroactive compounds is required. Thus, we have developed a mass spectrometry imaging method to visualize and quantify the brain distribution of drugs with varying blood-brain barrier permeability. With this approach, we were able to determine blood-brain barrier transport of different drugs and define the drug distribution in very small brain structures (e.g., choroid plexus) due to the high spatial resolution provided. Simultaneously, we investigated the effect of drug-drug interactions by inhibiting the membrane transporter multidrug resistance 1 protein. We propose that the described approach can serve as a valuable analytical tool during the development of neuroactive drugs, as it can provide physiologically relevant information often neglected by traditional imaging technologies.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Loperamida/farmacocinética , Propranolol/farmacocinética , Espectrometria de Massas por Ionização por Electrospray/métodos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Interações Medicamentosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição TecidualRESUMO
Advances in mass spectrometry imaging that improve both spatial and mass resolution are resulting in increasingly larger data files that are difficult to handle with current software. We have developed a novel near-lossless compression method with data entropy reduction that reduces the file size significantly. The reduction in data size can be set at four different levels (coarse, medium, fine, and superfine) prior to running the data compression. This can be applied to spectra or spectrum-by-spectrum, or it can be applied to transpose arrays or array-by-array, to efficiently read the data without decompressing the whole data set. The results show that a compression ratio of up to 5.9:1 was achieved for data from commercial mass spectrometry software programs and 55:1 for data from our in-house developed msIQuant program. Comparing the average signals from regions of interest, the maximum deviation was 0.2% between compressed and uncompressed data sets with coarse accuracy for the data entropy reduction. In addition, when accessing the compressed data by selecting a random m/ z value using msIQuant, the time to update an image on the computer screen was only slightly increased from 92 (±32) ms (uncompressed) to 114 (±13) ms (compressed). Furthermore, the compressed data can be stored on readily accessible servers for data evaluation without further data reprocessing. We have developed a space efficient, direct access data compression algorithm for mass spectrometry imaging, which can be used for various data-demanding mass spectrometry imaging applications.
RESUMO
We present a strategy for imaging of elements in biological tissues using laser ablation (LA) mass spectrometry (MS), which was compared to laser ablation inductively coupled plasma (LA-ICP) MS. Both methods were adopted for quantitative imaging of elements in mouse kidney, as well as traumatic brain injury model tissue sections. MS imaging (MSI) employing LA provides quantitative data by comparing signal abundances of sodium from tissues to those obtained by imaging quantitation calibration standards of the target element applied to adjacent control tissue sections. LA-ICP MSI provided quantitative data for several essential elements in both brain and kidney tissue sections using a dried-droplet approach. Both methods were used to image a rat model of traumatic brain injury, revealing accumulations of sodium and calcium in the impact area and its peripheral regions. LA MSI is shown to be a viable option for quantitative imaging of specific elements in biological tissue sections.
Assuntos
Lesões Encefálicas Traumáticas/diagnóstico por imagem , Terapia a Laser/métodos , Espectrometria de Massas/métodos , Animais , Lesões Encefálicas Traumáticas/metabolismo , Cálcio/isolamento & purificação , Cálcio/metabolismo , Humanos , Rim/diagnóstico por imagem , Camundongos , Ratos , Sódio/isolamento & purificação , Sódio/metabolismoRESUMO
With neurological processes involving multiple neurotransmitters and neuromodulators, it is important to have the ability to directly map and quantify multiple signaling molecules simultaneously in a single analysis. By utilizing a molecular-specific approach, namely desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we demonstrated that the technique can be used to image multiple neurotransmitters and their metabolites (dopamine, dihydroxyphenylacetic acid, 3-methoxytyramine, serotonin, glutamate, glutamine, aspartate, γ-aminobutyric acid, adenosine) as well as neuroactive drugs (amphetamine, sibutramine, fluvoxamine) and drug metabolites in situ directly in brain tissue sections. The use of both positive and negative ionization modes increased the number of identified molecular targets. Chemical derivatization by charge-tagging the primary amines of molecules significantly increased the sensitivity, enabling the detection of low abundant neurotransmitters and other neuroactive substances previously undetectable by MSI. The sensitivity of the imaging approach of neurochemicals has a great potential in many diverse applications in fields such as neuroscience, pharmacology, drug discovery, neurochemistry, and medicine.
Assuntos
Algoritmos , Encéfalo/metabolismo , Imagem Molecular/métodos , Neurotransmissores/metabolismo , Psicotrópicos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Encéfalo/anatomia & histologia , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Processamento de Sinais Assistido por Computador , Distribuição TecidualRESUMO
This paper presents msIQuant, a novel instrument- and manufacturer-independent quantitative mass spectrometry imaging software suite that uses the standardized open access data format imzML. Its data processing structure enables rapid image display and the analysis of very large data sets (>50 GB) without any data reduction. In addition, msIQuant provides many tools for image visualization including multiple interpolation methods, low intensity transparency display, and image fusion. It also has a quantitation function that automatically generates calibration standard curves from series of standards that can be used to determine the concentrations of specific analytes. Regions-of-interest in a tissue section can be analyzed based on a number of quantities including the number of pixels, average intensity, standard deviation of intensity, and median and quartile intensities. Moreover, the suite's export functions enable simplified postprocessing of data and report creation. We demonstrate its potential through several applications including the quantitation of small molecules such as drugs and neurotransmitters. The msIQuant suite is a powerful tool for accessing and evaluating very large data sets, quantifying drugs and endogenous compounds in tissue areas of interest, and for processing mass spectra and images.
Assuntos
Conjuntos de Dados como Assunto , Espectrometria de Massas/métodos , Software , Animais , Encéfalo , Pulmão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
The limit of detection of low-molecular weight compounds in tissue sections, analyzed by matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), was significantly improved by employing sample washing using a pH-controlled buffer solution. The pH of the washing solutions were set at values whereby the target analytes would have low solubility. Washing the tissue sections in the buffered solution resulted in removal of endogenous soluble ionization-suppressing compounds and salts, while the target compound remained in situ with minor or no delocalization during the buffered washing procedure. Two pharmaceutical compounds (cimetidine and imipramine) and one new protease inhibitor compound were successfully used to evaluate the feasibility of the pH-controlled tissue washing protocol for MALDI-MSI. Enhancement in signal-to-noise ratio was achieved by a factor of up to 10.
Assuntos
Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Encéfalo/metabolismo , Cimetidina/análise , Cimetidina/isolamento & purificação , Concentração de Íons de Hidrogênio , Imipramina/análise , Imipramina/isolamento & purificação , Masculino , Camundongos , Preparações Farmacêuticas/isolamento & purificação , Ratos , Ratos WistarRESUMO
D(4)-α-Cyano-4-hydroxycinnamic acid (D(4)-CHCA) has been synthesized for use as a matrix for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) and MALDI-MS imaging (MSI) of small molecule drugs and endogenous compounds. MALDI-MS analysis of small molecules has historically been hindered by interference from matrix ion clusters and fragment peaks that mask signals of low molecular weight compounds of interest. By using D(4)-CHCA, the cluster and fragment peaks of CHCA, the most common matrix for analysis of small molecules, are shifted by + 4, + 8 and + 12 Da, which expose signals across areas of the previously concealed low mass range. Here, obscured MALDI-MS signals of a synthetic small molecule pharmaceutical, a naturally occurring isoquinoline alkaloid, and endogenous compounds including the neurotransmitter acetylcholine have been unmasked and imaged directly from biological tissue sections.
Assuntos
Ácidos Cumáricos/química , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetilcolina/metabolismo , Animais , Berberina/metabolismo , Encéfalo/metabolismo , Pulmão/metabolismo , Masculino , Camundongos , Peso Molecular , RatosRESUMO
The new Ru complex 8 containing the bio-inspired ligand 7 was successfully synthesized and characterized. Complex 8 efficiently catalyzes water oxidation using Ce(IV) and Ru(III) as chemical oxidants. More importantly, this complex has a sufficiently low overpotential to utilize ruthenium polypyridyl-type complexes as photosensitizers.
Assuntos
Ligantes , Luz , Rutênio/química , Água/química , Cério/química , Estrutura Molecular , Oxirredução , Oxigênio/química , FotoquímicaRESUMO
Downscaled analytical tools for sample preparation have offered benefits such as higher throughput, easier automation and lower sample/reagent consumption. Microfluidic electrocapture, which is a newly developed sample preparation/manipulation system, uses an electric field to trap and separate charged species without using any solid sorbent. The feasibility of using microfluidic electrocapture is reported for separation, clean-up, concentration, microreactions and complexation studies of proteins, peptides and other biologically important biomolecules. The instrumentation and applications of microfluidic electrocapture are reviewed and an overview is provided of future perspectives offered by the current and envisaged platforms.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/tendências , Animais , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/métodos , Peptídeos/análise , Proteínas/análiseRESUMO
BACKGROUND: Monoamine oxidase inhibitors (MAOIs) exert therapeutic actions by elevating extracellular levels of monoamines in the brain. Irreversible MAOIs cause serious hypertensive crises owing to peripheral accumulation of tyramine, but the role of tyramine in the central effects of MAOIs remains elusive, an issue addressed herein. To achieve robust inhibition of MAOA/B, the clinically used antidepressant tranylcypromine (TCP) was employed. METHODS: Behavioral, histological, mass spectrometry imaging, and biosensor-mediated measures of glutamate were conducted with MAOIs in wild-type and TAAR1-knockout (KO) mice. RESULTS: Both antidepressant and locomotion responses to TCP were enhanced in TAAR1-KO mice. A recently developed fluoromethylpyridinium-based mass spectrometry imaging method revealed robust accumulation of striatal tyramine on TCP administration. Furthermore, tyramine accumulation was higher in TAAR1-KO versus wild-type mice, suggesting a negative feedback mechanism for TAAR1 in sensing tyramine levels. Combined histoenzymological and immunohistological studies revealed hitherto unknown TAAR1 localization in brain areas projecting to the substantia nigra/ventral tegmental area. Using an enzyme-based biosensor technology, we found that both TCP and tyramine reduced glutamate release in the substantia nigra in wild-type but not in TAAR1-KO mice. Moreover, glutamate measures in freely moving animals treated with TCP demonstrated that TAAR1 prevents glutamate accumulation in the substantia nigra during hyperlocomotive states. CONCLUSIONS: These observations suggest that tyramine, in interaction with glutamate, is involved in centrally mediated behavioral, transcriptional, and neurochemical effects of MAOIs.
Assuntos
Ácido Glutâmico , Inibidores da Monoaminoxidase , Receptores Acoplados a Proteínas G/fisiologia , Tiramina , Animais , Camundongos , Camundongos Knockout , Monoaminoxidase , Inibidores da Monoaminoxidase/farmacologiaRESUMO
A simple platform for combining solid phase extraction (SPE) and surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) of extracted analytes, using disks prepared by embedding graphitized carbon black (GCB-4) particles in a network of polytetrafluoroethylene (PTFE), is presented. The system provides a convenient approach for rapid SALDI-MS screening of substances in aqueous samples, which can be followed by robust quantitative and/or structural analyses by liquid chromatography (LC)/MS/MS of positive samples. The extraction discs are easily transferred between gaskets where the sample extraction and desorption of selected samples is performed and the mass spectrometer. The SPE and SALDI properties of the new GCB-4 disc have been characterized for 15 pesticides with varying chemical properties, and the screening strategy has been applied to the analysis of pesticides in agricultural drainage water. Atrazine and atrazine-desethyl-2-hydroxy were detected in the sampled water by SALDI-MS screening and subsequently confirmed and quantified using LC/MS/MS.
Assuntos
Atrazina/química , Praguicidas/química , Fuligem/química , Água/química , Espectrometria de Massas/métodos , Politetrafluoretileno/química , Extração em Fase Sólida/métodos , Poluentes Químicos da Água/químicaAssuntos
Descoberta de Drogas/métodos , Espectrometria de Massas/métodos , Preparações Farmacêuticas/análise , Farmacocinética , Animais , Descoberta de Drogas/instrumentação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Humanos , Espectrometria de Massas/instrumentação , Preparações Farmacêuticas/metabolismo , Testes de Toxicidade/instrumentação , Testes de Toxicidade/métodosRESUMO
Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain-Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-A crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.
Assuntos
Proteínas de Ligação a Ácido Graxo/química , Ácidos Graxos/química , Espectrometria de Massas/métodos , Proteína P2 de Mielina/química , Sequência de Aminoácidos , Animais , Cristalização , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Humanos , Conformação Molecular , Dados de Sequência Molecular , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Ligação Proteica , Alinhamento de Sequência , SuínosRESUMO
Quaternary protoberberine alkaloids belong to a pharmaceutically important class of isoquinoline alkaloids associated with bactericidal, fungicidal, insecticidal and antiviral activities. As traditional medicine gains wider acceptance, quick and robust analytical methods for the screening and analysis of plants containing these compounds attract considerable interest. Thin-layer chromatography (TLC) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful technique but suffers from dilution of the TLC bands resulting in decreased sensitivity and masking of signals in the low-mass region both due to addition of matrix. This study integrates for the first time conventional silica gel TLC and laser desorption ionization mass spectrometry (LDI-MS) thus eliminating the need for any external matrix. Successful separation of berberine (R(f) = 0.56) and palmatine (R(f) = 0.46) from Berberis barandana including their identification by MS are demonstrated. Furthermore, a robust electrospray ionization (ESI)-MS method utilizing residual sample from TLC for quantification of berberine applying selected reaction monitoring and standard addition method is presented. The amount of berberine in the plant root prepared for the study was determined to be 0.70% (w/w).
Assuntos
Alcaloides de Berberina/isolamento & purificação , Berberina/isolamento & purificação , Berberis/química , Cromatografia em Camada Fina/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Berberina/química , Alcaloides de Berberina/química , Extratos Vegetais/química , Raízes de Plantas/química , Sensibilidade e EspecificidadeRESUMO
Matrix-assisted laser desorption/ionisation (MALDI) of small molecules is challenging and in most cases impossible due to interferences from matrix ions precluding analysis of molecules <300-500 Da. A common matrix such as ferulic acid belongs to an important class of compounds associated with antioxidant activity. If the shared phenolic structure is related to the propensity as an active MALDI matrix then it follows that direct laser desorption/ionisation should be possible for polyphenols. Indeed matrix-less laser desorption/ionisation mass spectrometry is achieved whereby the analyte functions as a matrix and was used to monitor low molecular weight compounds in wine samples. Sensitivity ranging from 0.12-87 pmol/spot was achieved for eight phenolic acids (4-coumaric, 4-hydroxybenzoic, caffeic, ferulic, gallic, protocatechuic, syringic, vanillic) and 0.02 pmol/spot for trans-resveratrol. Additionally, 4-coumaric, 4-hydroxybenzoic, caffeic, ferulic, gallic, syringic, vanillic acids and trans-resveratrol were identified in wine samples using accurate mass measurements consistent with reported profiles based on liquid chromatography (LC)/MS. Minimal sample pre-treatment make the technique potentially appropriate for fingerprinting, screening and quality control of wine samples.
Assuntos
Flavonoides/química , Fenóis/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Vinho/análise , Polifenóis , Sensibilidade e EspecificidadeRESUMO
We compare (29)Si magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra from the two modifications of silicon nitride, alpha-Si(3)N(4) and beta-Si(3)N(4), with that of a fully ((29)Si, (15)N)-enriched sample (29)Si(3)(15)N(4), as well as (15)N NMR spectra of Si(3)(15)N(4) (having (29)Si at natural abundance) and (29)Si(3)(15)N(4). We show that the (15)N NMR peak-widths from the latter are dominated by J((29)Si-(15)N) through-bond interactions, leading to significantly broader NMR signals compared to those of Si(3)(15)N(4). By fitting calculated (29)Si NMR spectra to experimental ones, we obtained an estimated coupling constant J((29)Si-(15)N) of 20Hz. We provide (29)Si spin-lattice (T(1)) relaxation data for the (29)Si(3)(15)N(4) sample and chemical shift anisotropy results for the (29)Si site of beta-Si(3)N(4). Various factors potentially contributing to the (29)Si and (15)N NMR peak-widths of the various silicon nitride specimens are discussed. We also provide powder X-ray diffraction (XRD) and mass spectrometry data of the samples.
RESUMO
The neurotransmitter of the cholinergic system, acetylcholine plays a major role in the brain's cognitive function and is involved in neurodegenerative disorders. Here, we present age-related alterations of acetylcholine levels after administration of the acetylcholinesterase inhibitor drug tacrine in normal mice. Using a quantitative, robust and molecular-specific mass spectrometry imaging method we found that tacrine administration significantly raised acetylcholine levels in most areas of sectioned mice brains, inter alia the striatum, hippocampus and cortical areas. However, acetylcholine levels in retrosplenial cortex were significantly lower in 14-month-old than in 12-week-old animals following its administration, indicating that normal aging affects the cholinergic system's responsivity. This small brain region is interconnected with an array of brain networks and is involved in numerous cognitive tasks. Simultaneous visualization of distributions of tacrine and its hydroxylated metabolites in the brain revealed a significant decrease in levels of the metabolites in the 14-month-old mice. The results highlight strengths of the imaging technique to simultaneously investigate multiple molecular species and the drug-target effects in specific regions of the brain. The proposed approach has high potential in studies of neuropathological conditions and responses to neuroactive treatments.
Assuntos
Acetilcolina/metabolismo , Envelhecimento/metabolismo , Córtex Cerebral/metabolismo , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/metabolismo , Tacrina/administração & dosagem , Acetilcolina/análise , Animais , Córtex Cerebral/química , Córtex Cerebral/efeitos dos fármacos , Inibidores da Colinesterase/análise , Masculino , Camundongos Endogâmicos C57BL , Imagem Molecular , Tacrina/análiseRESUMO
To isolate membrane-associated proteins, which play diverse structural, catalytic, and regulatory roles in cells, they are often initially solubilized in detergents. Although detergents are essential for purifying membrane proteins, they tend to interfere strongly with subsequent analyses. A microfluidic method is presented here that surmounts this problem, allowing well-resolved mass spectra of test membrane-associated polypeptides, and their complexes with ions and detergents, to be acquired. As a front-end module it allows access to other advanced mass spectrometric strategies to be utilized toward defining biomolecular interactions. This opens up a new avenue for studying complexation and analysis of membrane proteins of general importance.