RESUMO
We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex isobaric tags for relative and absolute quantification (iTRAQ) chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically pumped sheath-flow nanospray interface. This HPLC-cIEF-electrospray-tandem mass spectrometry (ESI-MS/MS) approach identified 835 protein groups and produced 2,329 unique peptides IDs. The biological duplicates were analyzed in parallel using conventional strong-cation exchange (SCX)-RPLC-ESI-MS/MS. The iTRAQ peptides were first separated into eight fractions using SCX. Each fraction was then analyzed by RPLC-ESI-MS/MS. The SCX-RPLC approach generated 1,369 protein groups and 3,494 unique peptide IDs. For protein quantitation, 96 and 198 differentially expressed proteins were obtained with RPLC-cIEF and SCX-RPLC, respectively. The combined set identified 231 proteins. Protein expression changes measured by RPLC-cEIF and SCX-RPLC were highly correlated.
Assuntos
Diferenciação Celular , Cromatografia de Fase Reversa/métodos , Focalização Isoelétrica/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Células PC12 , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas/isolamento & purificação , Proteínas/metabolismo , Ratos , Fatores de TempoRESUMO
Chemical cytometry employs modern analytical methods to study the differences in composition between single cells to better understand development, cellular differentiation, and disease. Metabolic cytometry is a form of chemical cytometry wherein cells are incubated with and allowed to metabolize fluorescently labeled small molecules. Capillary electrophoresis with laser-induced fluorescence detection is then used to characterize the extent of metabolism at the single cell level. To date, all metabolic cytometry experiments have used conventional two-dimensional cell cultures. HCT 116 spheroids are a three-dimensional cell culture system, morphologically and phenotypically similar to tumors. Here, intact HCT 116 multicellular spheroids were simultaneously incubated with three fluorescently labeled glycosphingolipid substrates, GM3-BODIPY-FL, GM1-BODIPY-TMR, and lactosylceramide-BODIPY-650/665. These substrates are spectrally distinct, and their use allows the simultaneous probing of metabolism at three different points in the glycolipid metabolic cascade. Beginning with intact spheroids, a serial trypsinization and trituration procedure was used to isolate single cells from spatially distinct regions of the spheroid. Cells from the distinct regions showed unique metabolic patterns. Treatment with the lysosomal inhibitor and potential chemotherapeutic chloroquine consistently decreased catabolism for all substrates. Nearly 200 cells were taken for analysis. Principal component analysis with a multivariate measure of precision was used to quantify cell-to-cell variability in glycosphingolipid metabolism as a function of cellular localization and chloroquine treatment. While cells from different regions exhibited differences in metabolism, the heterogeneity in metabolism did not differ significantly across the experimental conditions.
Assuntos
Metaboloma , Neoplasias/metabolismo , Análise de Célula Única , Esferoides Celulares/metabolismo , Cloroquina/farmacologia , Citometria de Fluxo , Glicoesfingolipídeos/metabolismo , Humanos , Metaboloma/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Análise de Componente Principal , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Células Tumorais CultivadasRESUMO
Over the last several decades, fast-scan cyclic voltammetry (FSCV) has proved to be a valuable analytical tool for the real-time measurement of neurotransmitter dynamics in vitro and in vivo. Indeed, FSCV has found application in a wide variety of disciplines including electrochemistry, neurobiology, and behavioral psychology. The maturation of FSCV as an in vivo technique led users to pose increasingly complex questions that require a more sophisticated experimental design. To accommodate recent and future advances in FSCV application, our lab has developed High Definition Cyclic Voltammetry (HDCV). HDCV is an electrochemical software suite that includes data acquisition and analysis programs. The data collection program delivers greater experimental flexibility and better user feedback through live displays. It supports experiments involving multiple electrodes with customized waveforms. It is compatible with transistor-transistor logic-based systems that are used for monitoring animal behavior, and it enables simultaneous recording of electrochemical and electrophysiological data. HDCV analysis streamlines data processing with superior filtering options, seamlessly manages behavioral events, and integrates chemometric processing. Furthermore, analysis is capable of handling single files collected over extended periods of time, allowing the user to consider biological events on both subsecond and multiminute time scales. Here we describe and demonstrate the utility of HDCV for in vivo experiments.
Assuntos
Técnicas Eletroquímicas/métodos , Software , Animais , HumanosRESUMO
A capillary electrophoresis system with an ultrasensitive three-color laser-induced fluorescence detector was constructed for the simultaneous measurement of glycosphingolipids conjugated with a family of BODIPY fluorophores. The compounds were separated by capillary electrophoresis and detected by laser-induced fluorescence excited within a sheath-flow cuvette. Diode-pumped solid-state lasers operating at 473 nm and 532 nm, and a diode laser operating at 633 nm were used to excite glycosphingolipids tagged with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 fluorophores. Detection limits were 34 ± 1 molecules, 67 ± 7 molecules, and 36 ± 13 molecules of BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled glycosphingolipids. Separation efficiencies were typically one million theoretical plates. To test the ability of the system to analyze cellular contents in an in vitro biological model, differentiated PC12 cells were co-incubated with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled lactosylceramide substrates. Cells were homogenized. The metabolic products originating from the glycosphingolipid substrates were simultaneously analyzed using the system.
Assuntos
Eletroforese Capilar/métodos , Glicoesfingolipídeos/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Compostos de Boro/química , Cor , Glicoesfingolipídeos/química , Lasers , Células PC12 , Ratos , Espectrometria de Fluorescência/instrumentaçãoRESUMO
Mesolimbic dopamine neurons fire in both tonic and phasic modes resulting in detectable extracellular levels of dopamine in the nucleus accumbens (NAc). In the past, different techniques have targeted dopamine levels in the NAc to establish a basal concentration. In this study, we used in vivo fast scan cyclic voltammetry (FSCV) in the NAc of awake, freely moving rats. The experiments were primarily designed to capture changes in dopamine caused by phasic firing - that is, the measurement of dopamine 'transients'. These FSCV measurements revealed for the first time that spontaneous dopamine transients constitute a major component of extracellular dopamine levels in the NAc. A series of experiments were designed to probe regulation of extracellular dopamine. Lidocaine was infused into the ventral tegmental area, the site of dopamine cell bodies, to arrest neuronal firing. While there was virtually no instantaneous change in dopamine concentration, longer sampling revealed a decrease in dopamine transients and a time-averaged decrease in the extracellular level. Dopamine transporter inhibition using intravenous GBR12909 injections increased extracellular dopamine levels changing both frequency and size of dopamine transients in the NAc. To further unmask the mechanics governing extracellular dopamine levels we used intravenous injection of the vesicular monoamine transporter (VMAT2) inhibitor, tetrabenazine, to deplete dopamine storage and increase cytoplasmic dopamine in the nerve terminals. Tetrabenazine almost abolished phasic dopamine release but increased extracellular dopamine to â¼500 nM, presumably by inducing reverse transport by dopamine transporter (DAT). Taken together, data presented here show that average extracellular dopamine in the NAc is low (20-30 nM) and largely arises from phasic dopamine transients.
Assuntos
Dopamina/metabolismo , Espaço Extracelular/metabolismo , Núcleo Accumbens/metabolismo , Anestésicos Locais/administração & dosagem , Anestésicos Locais/farmacologia , Animais , Dopamina/fisiologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Fenômenos Eletrofisiológicos , Lidocaína/administração & dosagem , Lidocaína/farmacologia , Masculino , Microdiálise , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Tetrabenazina/farmacologia , Área Tegmentar Ventral , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Proteínas Vesiculares de Transporte de Neurotransmissores/antagonistas & inibidores , Proteínas Vesiculares de Transporte de Neurotransmissores/metabolismoRESUMO
Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.
Assuntos
Eletroforese Capilar/métodos , Glicoesfingolipídeos/metabolismo , Neurônios/metabolismo , Animais , Compostos de Boro/análise , Compostos de Boro/metabolismo , Encéfalo/citologia , Células Cultivadas , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Fluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Glicoesfingolipídeos/análise , Lactosilceramidas/análise , Lactosilceramidas/metabolismo , Limite de Detecção , Redes e Vias Metabólicas , Microscopia de Fluorescência , Ratos , Rodaminas/análise , Rodaminas/metabolismoRESUMO
Cell-to-cell heterogeneity in ganglioside catabolism was determined by profiling fluorescent tetramethylrhodamine-labeled GM1 (TMR-GM1) breakdown in individual primary neurons and glia from the rat cerebellum. Cells isolated from 5 to 6 day old rat cerebella were cultured for 7 days, and then incubated for 14 h with TMR-GM1. Intact cells were recovered from cultures by mild proteolysis, paraformaldehyde fixed, and subjected to single cell analysis. Individual cells were captured in a capillary, lysed, and the released single-cell contents analyzed by capillary electrophoresis with quantitative laser-induced fluorescent detection of metabolites. Non-neuronal cells on average took up much more exogenous TMR-GM1 than neuronal cells, and catabolized it more extensively. After 14 h of incubation, non-neuronal cells retained only 14% of the TMR products as GM1 and GM2, compared to >50% for neurons. On average, non-neuronal cells contained 74% of TMR-labeled product as TMR-ceramide, compared to only 42% for neurons. Non-neuronal cells retained seven times as much TMR-GM3 (7%) compared to neuronal cells (1%). To confirm the observed single cell metabolomics, we lysed and compared TMR-GM1 catabolic profiles from mixed neuron/glial cell cultures and from cultures depleted of non-neuronal cells by treatment with the antimitotic agent cytosine arabinoside. The lysed culture catabolic profiles were consistent with the average profiles of single neurons and glia. We conclude that the ultrasensitive analytic methods described accurately reflect single cell ganglioside catabolism in different cell populations from the brain.
Assuntos
Cerebelo/citologia , Gangliosídeo G(M1)/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Análise de Célula Única/métodos , Animais , Células Cultivadas , Gangliosídeo G(M3)/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Fast-scan cyclic voltammetry (FSCV) with carbon-fiber microelectrodes has been successfully used to detect catecholamine release in vivo. Generally, waveforms with anodic voltage limits of 1.0 or 1.3 V (vs Ag/AgCl) are used for detection. The 1.0 V excursion provides good temporal resolution but suffers from a lack of sensitivity. The 1.3 V excursion increases sensitivity but also increases response time, which can blur the detection of neurochemical events. Here, the scan rate was increased to improve the sensitivity of the 1.0 V excursion while maintaining the rapid temporal response. However, increasing scan rate increases both the desired faradaic current response and the already large charging current associated with the voltage sweep. Analog background subtraction was used to prevent the analog-to-digital converter from saturating from the high currents generated with increasing scan rate by neutralizing some of the charging current. In vitro results with the 1.0 V waveform showed approximately a 4-fold increase in signal-to-noise ratio with maintenance of the desired faster response time by increasing scan rate up to 2400 V/s. In vivo, stable stimulated release was detected with an approximate 4-fold increase in peak current. The scan rate of the 1.3 V waveform was also increased, but the signal was unstable with time in vitro and in vivo. Adapting the 1.3 V triangular wave into a sawhorse design prevented signal decay and increased the faradaic response. The use of the 1.3 V sawhorse waveform decreased the detection limit of dopamine with FSCV to 0.96 ± 0.08 nM in vitro and showed improved performance in vivo without affecting the neuronal environment. Electron microscopy showed dopamine sensitivity is in a quasi-steady state with carbon-fiber microelectrodes scanned to potentials above 1.0 V.
Assuntos
Dopamina/metabolismo , Eletroquímica/métodos , Animais , Carbono/química , Fibra de Carbono , Condutividade Elétrica , Eletroquímica/instrumentação , Masculino , Microeletrodos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Propriedades de Superfície , Fatores de TempoRESUMO
Principal component regression has been used in the past to separate current contributions from different neuromodulators measured with in vivo fast-scan cyclic voltammetry. Traditionally, a percent cumulative variance approach has been used to determine the rank of the training set voltammetric matrix during model development; however, this approach suffers from several disadvantages including the use of arbitrary percentages and the requirement of extreme precision of training sets. Here, we propose that Malinowski's F-test, a method based on a statistical analysis of the variance contained within the training set, can be used to improve factor selection for the analysis of in vivo fast-scan cyclic voltammetric data. These two methods of rank estimation were compared at all steps in the calibration protocol including the number of principal components retained, overall noise levels, model validation as determined using a residual analysis procedure, and predicted concentration information. By analyzing 119 training sets from two different laboratories amassed over several years, we were able to gain insight into the heterogeneity of in vivo fast-scan cyclic voltammetric data and study how differences in factor selection propagate throughout the entire principal component regression analysis procedure. Visualizing cyclic voltammetric representations of the data contained in the retained and discarded principal components showed that using Malinowski's F-test for rank estimation of in vivo training sets allowed for noise to be more accurately removed. Malinowski's F-test also improved the robustness of our criterion for judging multivariate model validity, even though signal-to-noise ratios of the data varied. In addition, pH change was the majority noise carrier of in vivo training sets while dopamine prediction was more sensitive to noise.
Assuntos
Técnicas Eletroquímicas/métodos , Animais , Dopamina/análise , Masculino , Análise Multivariada , Análise de Componente Principal , Ratos , Ratos Sprague-DawleyRESUMO
Transient local pH changes in the brain are important markers of neural activity that can be used to follow metabolic processes that underlie the biological basis of behavior, learning and memory. There are few methods that can measure pH fluctuations with sufficient time resolution in freely moving animals. Previously, fast-scan cyclic voltammetry at carbon-fiber microelectrodes was used for the measurement of such pH transients. However, the origin of the potential dependent current in the cyclic voltammograms for pH changes recorded in vivo was unclear. The current work explored the nature of these peaks and established the origin for some of them. A peak relating to the capacitive nature of the pH CV was identified. Adsorption of electrochemically inert species, such as aromatic amines and calcium could suppress this peak, and is the origin for inconsistencies regarding in vivo and in vitro data. Also, we identified an extra peak in the in vivo pH CV relating to the presence of 3,4-dihydroxyacetic acid (DOPAC) in the brain extracellular fluid. To evaluate the in vivo performance of the carbon-fiber sensor, carbon dioxide inhalation by an anesthetized rat was used to induce brain acidosis induced by hypercapnia. Hypercapnia is demonstrated to be a useful tool to induce robust in vivo pH changes, allowing confirmation of the pH signal observed with FSCV.
Assuntos
Encéfalo/metabolismo , Carbono/química , Técnicas Eletroquímicas/métodos , Ácido 3,4-Di-Hidroxifenilacético/química , Animais , Análise de Injeção de Fluxo , Concentração de Íons de Hidrogênio , Masculino , Microeletrodos , Ratos , Ratos Sprague-DawleyRESUMO
Electrode fouling decreases sensitivity and can be a substantial limitation in electrochemical experiments. In this work we describe an electrochemical procedure that constantly renews the surface of a carbon microelectrode using periodic triangle voltage excursions to an extended anodic potential at a scan rate of 400 V s(-1). This methodology allows for the regeneration of an electrochemically active surface and restores electrode sensitivity degraded by irreversible adsorption of chemical species. We show that repeated voltammetric sweeps to moderate potentials in aqueous solution causes oxidative etching of carbon thereby constantly renewing the electrochemically active surface. Oxidative etching was established by tracking surface-localized fluorine atoms with XPS, by monitoring changes in carbon surface morphology with AFM on pyrolyzed photoresist films, and also by optical and electron microscopy. The use of waveforms with extended anodic potentials showed substantial increases in sensitivity toward the detection of catechols. This enhancement arose from the adsorption of the catechol moiety that could be maintained with a constant regeneration of the electrode surface. We also demonstrate that application of the extended waveform could restore the sensitivity of carbon microelectrodes diminished by irreversible adsorption (electrode fouling) of byproducts resulting from the electrooxidation and polymerization of tyramine. Overall, this work brings new insight into the factors that affect electrochemical processes at carbon electrodes and provides a simple method to remove or reduce fouling problems associated with many electrochemical experiments.
Assuntos
Carbono/química , Microeletrodos , Microscopia de Força Atômica , Propriedades de SuperfícieRESUMO
5-Hydroxytryptamine (5-HT) is an important molecule in the brain that is implicated in mood and emotional processes. In vivo, its dynamic release and uptake kinetics are poorly understood due to a lack of analytical techniques for its rapid measurement. Whereas fast-scan cyclic voltammetry with carbon fiber microelectrodes is used frequently to monitor subsecond dopamine release in freely moving and anesthetized rats, the electrooxidation of 5-HT forms products that quickly polymerize and irreversibly coat the carbon electrode surface. Previously described modifications of the electrochemical waveform allow stable and sensitive 5-HT measurements in mammalian tissue slice preparations and in the brain of fruit fly larvae. For in vivo applications in mammals, however, the problem of electrode deterioration persists. We identify the root of this problem to be fouling by extracellular metabolites such as 5-hydoxyindole acetic acid (5-HIAA), which is present in 200-1000 times the concentration of 5-HT and displays similar electrochemical properties, including filming of the electrode surface. To impede access of the 5-HIAA to the electrode surface, a thin layer of Nafion, a cation exchange polymer, has been electrodeposited onto cylindrical carbon-fiber microelectrodes. The presence of the Nafion film was confirmed with environmental scanning electron microscopy and was demonstrated by the diminution of the voltammetric signals for 5-HIAA as well as other common anionic species. The modified microelectrodes also display increased sensitivity to 5-HT, yielding a characteristic cyclic voltammogram that is easily distinguishable from other common electroactive brain species. The thickness of the Nafion coating and a diffusion coefficient (D) in the film for 5-HT were evaluated by measuring permeation through Nafion. In vivo, we used physiological, anatomical, and pharmacological evidence to validate the signal as 5-HT. Using Nafion-modified microelectrodes, we present the first endogenous recording of 5-HT in the mammalian brain.
Assuntos
Encéfalo/metabolismo , Técnicas de Química Analítica/métodos , Polímeros de Fluorcarboneto/química , Microeletrodos , Serotonina/metabolismo , Animais , Química Encefálica , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Serotonina/análiseRESUMO
Data analysis is an essential tenet of analytical chemistry, extending the possible information obtained from the measurement of chemical phenomena. Chemometric methods have grown considerably in recent years, but their wide use is hindered because some still consider them too complicated. The purpose of this review is to describe a multivariate chemometric method, principal component regression, in a simple manner from the point of view of an analytical chemist, to demonstrate the need for proper quality-control (QC) measures in multivariate analysis and to advocate the use of residuals as a proper QC method.
RESUMO
Fast-scan cyclic voltammetry has been used in a variety of applications and has been shown to be especially useful to monitor chemical fluctuations of neurotransmitters such as dopamine within the mammalian brain. A major limitation of this procedure, however, is the large amplitude of the background current relative to the currents for the solution species of interest. Furthermore, the background tends to drift, and this drift limits the use of digital background subtraction techniques to intervals less than 90 s before distortion of dopamine signals occurs. To minimize the impact of the background, a procedure termed analog background subtraction is reported here. The background is recorded, and its inverse is played back to the current transducer during data acquisition so that it cancels the background in subsequent scans. Background drift still occurs and is recorded, but its magnitude is small compared to the original background. This approach has two advantages. First it allows the use of higher gains in the current transducer, minimizing quantization noise. Second, because the background amplitude is greatly reduced, principal component regression could be used to separate the contributions from drift, dopamine, and pH when appropriate calibrations were performed. We demonstrate the use of this approach with several applications. First, transient dopamine fluctuations were monitored for 15 min in a flowing injection apparatus. Second, evoked release of dopamine was monitored for a similar period in the brain of an anesthetized rat. Third, dopamine was monitored in the brain of freely moving rats over a 30 min interval. By analyzing the fluctuations in each resolved component, we were able to show that cocaine causes significant fluctuations in dopamine concentration in the brain while those for the background and pH remain unchanged from their predrug value.
Assuntos
Dopamina/análise , Conversão Análogo-Digital , Anestesia , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Carbono/química , Fibra de Carbono , Eletroquímica , Masculino , Microeletrodos , Análise de Componente Principal , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Transdutores , CaminhadaRESUMO
The in vitro intrinsic clearances (CL(int)) for the metabolism of p-methoxymethamphetamine (PMMA) and fluoxetine by the CYP2D6 enzyme were calculated using a steady-state (SS) approach and a new general enzyme (GE) method, which measures the formation of product and the depletion of substrate as a function of time. For PMMA, the SS experiment resulted in a CL(int) of 2.7+/-0.2 microL pmol 2D6(-1)min(-1) and the GE experiment resulted in a CL(int) of 3.0+/-0.6 microL pmol 2D6(-1)min(-1). For fluoxetine, the SS experiment resulted in a CL(int) of 0.33+/-0.17 microL pmol 2D6(-1)min(-1) and the GE experiment resulted in a CL(int) of 0.188+/-0.013 microL pmol 2D6(-1)min(-1). We used two kinetic modeling techniques that can accommodate atypical kinetic models. We also show that the addition of fluoxetine results in a 10-fold decrease in the observed intrinsic clearance of PMMA, confirming that fluoxetine is a potent inhibitor of the liver enzyme CYP2D6.
Assuntos
Citocromo P-450 CYP2D6/metabolismo , Metanfetamina/análogos & derivados , Cromatografia Líquida/métodos , Fluoxetina/farmacologia , Cinética , Metanfetamina/antagonistas & inibidores , Metanfetamina/farmacocinética , Padrões de Referência , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
As imaging mass spectrometry (IMS) has grown in popularity in recent years, the applications of this technique have become increasingly diverse. Currently there is a need for sophisticated data processing strategies that maximize the information gained from large IMS data sets. Traditional two-dimensional heat maps of single ions generated in IMS experiments lack analytical detail, yet manual analysis of multiple peaks across hundreds of pixels within an entire image is time-consuming, tedious and subjective. Here, various chemometric methods were used to analyze data sets obtained by matrix-assisted laser desorption/ionization (MALDI) IMS of multicellular spheroids. HT-29 colon carcinoma multicellular spheroids are an excellent in vitro model system that mimic the three dimensional morphology of tumors in vivo. These data are especially challenging to process because, while different microenvironments exist, the cells are clonal which can result in strong similarities in the mass spectral profiles within the image. In this proof-of-concept study, a combination of principal component analysis (PCA), clustering methods, and linear discriminant analysis was used to identify unique spectral features present in spatially heterogeneous locations within the image. Overall, the application of these exploratory data analysis tools allowed for the isolation and detection of proteomic changes within IMS data sets in an easy, rapid, and unsupervised manner. Furthermore, a simplified, non-mathematical theoretical introduction to the techniques is provided in addition to full command routines within the MATLAB programming environment, allowing others to easily utilize and adapt this approach.
RESUMO
A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored. Aspiration of single differentiated PC12 cells suspended in a phosphate-buffered saline solution showed excessive peak dispersion, poor resolution, and peak efficiencies below 100,000 theoretical plates. Aspiration of single differentiated PC12 cells suspended in deionized water corrected peak dispersion. Average peak efficiencies ranged between 400,000 and 600,000 theoretical plates. Improved performance was due to the dilution of the high salt concentrations inside of single neuronal-like cells to produce field amplified sample stacking. Single cell separations showed the highest resolution when aspiration of single differentiated PC12 cells suspended in deionized water were separated using a running buffer of high ionic strength. The improvement in resolution allowed for the identification of analytes not previously detected in single cell metabolism studies.
Assuntos
Diferenciação Celular , Eletroforese Capilar/métodos , Glicoesfingolipídeos/isolamento & purificação , Análise de Célula Única/métodos , Animais , Compostos de Boro/química , Soluções Tampão , Corantes Fluorescentes/química , Gangliosídeos/química , Gangliosídeos/metabolismo , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Estrutura Molecular , Concentração Osmolar , Células PC12 , Ratos , Reprodutibilidade dos Testes , Cloreto de Sódio/química , Água/químicaRESUMO
BACKGROUND: Traditionally, norepinephrine has been associated with stress responses, whereas dopamine has been associated with reward. Both of these catecholamines are found within the bed nucleus of the stria terminalis (BNST), a brain relay nucleus in the extended amygdala between cortical/limbic centers, and the hypothalamic-pituitary-adrenal axis. Despite this colocalization, little is known about subsecond catecholamine signaling in subregions of the BNST in response to salient stimuli. METHODS: Changes in extracellular catecholamine concentration in subregions of the BNST in response to salient stimuli were measured within the rat BNST with fast-scan cyclic voltammetry at carbon-fiber microelectrodes. RESULTS: A discrete subregional distribution of release events was observed for different catecholamines in this nucleus. In addition, rewarding and aversive tastants evoked inverse patterns of norepinephrine and dopamine release in the BNST. An aversive stimulus, quinine, activated noradrenergic signaling but inhibited dopaminergic signaling, whereas a palatable stimulus, sucrose, inhibited norepinephrine while causing dopamine release. CONCLUSIONS: This reciprocal relationship, coupled with their different time courses, can provide integration of opposing hedonic states to influence response outputs appropriate for survival.
Assuntos
Dopamina/metabolismo , Vias Neurais/metabolismo , Norepinefrina/metabolismo , Recompensa , Núcleos Septais/metabolismo , Neurônios Adrenérgicos/metabolismo , Animais , Comportamento Animal/fisiologia , Técnicas Biossensoriais/métodos , Hormônio Liberador da Corticotropina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Ratos , Ratos Sprague-Dawley , Estresse Fisiológico/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Several glycosphingolipids were labeled with the fluorphore Bodipy-Fl and analyzed using capillary electrophoresis with laser-induced fluorescence detection. GM1-, LacCer-, and Cer-Bodipy-Fl were prepared through acylation using the N-hydroxysuccinimide ester of Bodipy-Fl. Several other glycosphingolipids including GT1a-, GD1a-, GM2-, GM3-, GD3-, and GlcCer-Bodipy-Fl were enzymatically synthesized. Micellar electrokinetic capillary chromatography with a TRIS/CHES/SDS/α-cyclodextrin buffer produced better separation than an established borate/deoxycholate/methyl-ß-cyclodextrin buffer. The nine Bodipy-Fl-labeled glycosphingolipid standards were separated in under 5 min, theoretical plate counts were between 640,000 and 740,000, and the limit of detection was approximately 3 pM or 240 ymol analyte injected onto the capillary.
Assuntos
Compostos de Boro/química , Cromatografia Capilar Eletrocinética Micelar/métodos , Eletroforese Capilar/métodos , Corantes Fluorescentes/química , Glicoesfingolipídeos/química , Boratos , Ácido Desoxicólico , Glicoesfingolipídeos/isolamento & purificação , Limite de Detecção , Dodecilsulfato de Sódio , Taurina/análogos & derivados , Trometamina , alfa-Ciclodextrinas , beta-CiclodextrinasRESUMO
Principal component regression is a multivariate data analysis approach routinely used to predict neurochemical concentrations from in vivo fast-scan cyclic voltammetry measurements. This mathematical procedure can rapidly be employed with present day computer programming languages. Here, we evaluate several methods that can be used to evaluate and improve multivariate concentration determination. The cyclic voltammetric representation of the calculated regression vector is shown to be a valuable tool in determining whether the calculated multivariate model is chemically appropriate. The use of Cook's distance successfully identified outliers contained within in vivo fast-scan cyclic voltammetry training sets. This work also presents the first direct interpretation of a residual color plot and demonstrated the effect of peak shifts on predicted dopamine concentrations. Finally, separate analyses of smaller increments of a single continuous measurement could not be concatenated without substantial error in the predicted neurochemical concentrations due to electrode drift. Taken together, these tools allow for the construction of more robust multivariate calibration models and provide the first approach to assess the predictive ability of a procedure that is inherently impossible to validate because of the lack of in vivo standards.