27 research outputs found

    Droplet Electrospray Ionization Mass Spectrometry for High Throughput Screening for Enzyme Inhibitors

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    High throughput screening (HTS) is important for identifying molecules with desired properties. Mass spectrometry (MS) is potentially powerful for label-free HTS due to its high sensitivity, speed, and resolution. Segmented flow, where samples are manipulated as droplets separated by an immiscible fluid, is an intriguing format for high throughput MS because it can be used to reliably and precisely manipulate nanoliter volumes and can be directly coupled to electrospray ionization (ESI) MS for rapid analysis. In this study, we describe a ā€œMS Plate Readerā€ that couples standard multiwell plate HTS workflow to droplet ESI-MS. The MS plate reader can reformat 3072 samples from eight 384-well plates into nanoliter droplets segmented by an immiscible oil at 4.5 samples/s and sequentially analyze them by MS at 2 samples/s. Using the system, a label-free screen for cathepsin B modulators against 1280 chemicals was completed in 45 min with a high <i>Z</i>-factor (>0.72) and no false positives (24 of 24 hits confirmed). The assay revealed 11 structures not previously linked to cathepsin inhibition. For even larger scale screening, reformatting and analysis could be conducted simultaneously, which would enable more than 145ā€‰000 samples to be analyzed in 1 day

    Western Blotting Using Microchip Electrophoresis Interfaced to a Protein Capture Membrane

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    Western blotting is a commonly used assay for proteins. Despite the utility of the method, it is also characterized by long analysis times, manual operation, and lack of established miniaturized counterpart. We report a new way to Western blot that addresses these limitations. In the method, sodium dodecyl sulfate (SDS)-protein complexes are separated by sieving electrophoresis in a microfluidic device or chip. The chip is interfaced to a moving membrane so that proteins are captured in discrete zones as they migrate from the chip. Separations of SDS-protein complexes in the molecular weight range of 11ā€“155 kDa were completed in 2 min with 4 Ɨ 10<sup>4</sup> theoretical plates at 460 V/cm. Migration time and peak area relative standard deviations were 3ā€“6% and 0.2%, respectively. Detection limit for actin was 0.7 nM. Assays for actin, AMP-kinase, carbonic anhydrase, and lysozyme are shown to demonstrate versatility of the method. Total analysis time including immunoassay was 22ā€“32 min for a single sample. Because processing membrane for immunoassay is the slow step of the assay, sequential injections from different reservoirs on the chip and capture in different tracks on the same membrane allow increased throughput. As a demonstration, 9 injections were collected on one membrane and analyzed in 43 min (āˆ¼5 min/sample). Further improvements in throughput are possible with more reservoirs or parallel channels

    Label Free Screening of Enzyme Inhibitors at Femtomole Scale Using Segmented Flow Electrospray Ionization Mass Spectrometry

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    Droplet-based microfluidics is an attractive platform for screening and optimizing chemical reactions. Using this approach, it is possible to reliably manipulate nanoliter volume samples and perform operations such as reagent addition with high precision, automation, and throughput. Most studies using droplet microfluidics have relied on optical techniques to detect the reaction; however, this requires engineering color or fluorescence change into the reaction being studied. In this work, we couple electrospray ionization mass spectrometry (ESI-MS) to nanoliter scale segmented flow reactions to enable direct (label-free) analysis of reaction products. The system is applied to a screen of inhibitors for cathepsin B. In this approach, solutions of test compounds (including three known inhibitors) are arranged as an array of nanoliter droplets in a tube segmented by perfluorodecalin. The samples are pumped through a series of tees to add enzyme, substrate (peptides), and quenchant. The resulting reaction mixtures are then infused into a metal-coated, fused silica ESI emitter for MS analysis. The system has potential for high-throughput as reagent addition steps are performed at 0.7 s per sample and ESI-MS at up to 1.2 s per sample. Carryover is inconsequential in the ESI emitter and between 2 and 9% per reagent addition depending on the tee utilized. The assay was reliable with a <i>Z</i>-factor of āˆ¼0.8. The method required 0.8 pmol of test compound, 1.6 pmol of substrate, and 5 fmol of enzyme per reaction. Segmented flow ESI-MS allows direct, label free screening of reactions at good throughput and ultralow sample consumption

    Microfluidic Chip with Integrated Electrophoretic Immunoassay for Investigating Cellā€“Cell Interactions

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    Microfluidics have been used to create ā€œbody-on-chipā€ systems to mimic in vivo cellular interactions with a high level of control. Most such systems rely on optical observation of cells as a readout. In this work we integrated a cellā€“cell interaction chip with online microchip electrophoresis immunoassay to monitor the effects of the interaction on protein secretion dynamics. The system was used to investigate the effects of adipocytes on insulin secretion. Chips were loaded with 190ā€Æ000 3T3-L1 adipocytes and a single islet of Langerhans in separate chambers. The chambers were perfused at 300ā€“600 nL/min so that adipocyte secretions flowed over the islets for 3 h. Adipocytes produced 80 Ī¼M of nonesterified fatty acids (NEFAs), a factor known to impact insulin secretion, at the islets. After perfusion, islets were challenged with a step change in glucose from 3 to 11 mM while monitoring insulin secretion at 8 s intervals by online immunoassay. Adipocyte treatment augmented insulin secretion by 6-fold compared to controls. The effect was far greater than comparable concentrations of NEFA applied to the islets demonstrating that adipocytes release multiple factors that can strongly potentiate insulin secretion. The experiments reveal that integration of chemical analysis with cellā€“cell interaction can provide valuable insights into cellular functions

    Microfabricated Sampling Probes for in Vivo Monitoring of Neurotransmitters

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    Microfabricated fluidic systems have emerged as a powerful approach for chemical analysis. Relatively unexplored is the use of microfabrication to create sampling probes. We have developed a sampling probe microfabricated in Si by bulk micromachining and lithography. The probe is 70 Ī¼m wide by 85 Ī¼m thick by 11 mm long and incorporates two buried channels that are 20 Ī¼m in diameter. The tip of the probe has two 20 Ī¼m holes where fluid is ejected or collected for sampling. Utility of the probe was demonstrated by sampling from the brain of live rats. For sampling, artificial cerebral spinal fluid was infused in through one channel at 50 nL/min while sample was withdrawn at the same flow rate from the other channel. Analysis of resulting fractions collected every 20 min from the striatum of rats by liquid chromatography with mass spectrometry demonstrated reliable detection of 17 neurotransmitters and metabolites. The small probe dimensions suggest it is less perturbing to tissue and can be used to sample smaller brain nuclei than larger sampling devices, such as microdialysis probes. This sampling probe may have other applications such as sampling from cells in culture. The use of microfabrication may also enable incorporation of electrodes for electrochemical or electrophysiological recording and other channels that enable more complex sample preparation on the device

    In Vivo Neurochemical Monitoring Using Benzoyl Chloride Derivatization and Liquid Chromatographyā€“Mass Spectrometry

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    In vivo neurochemical monitoring using microdialysis sampling is important in neuroscience because it allows correlation of neurotransmission with behavior, disease state, and drug concentrations in the intact brain. A significant limitation of current practice is that different assays are utilized for measuring each class of neurotransmitter. We present a high performance liquid chromatography (HPLC)ā€“tandem mass spectrometry method that utilizes benzoyl chloride for determination of the most common low molecular weight neurotransmitters and metabolites. In this method, 17 analytes were separated in 8 min. The limit of detection was 0.03ā€“0.2 nM for monoamine neurotransmitters, 0.05ā€“11 nM for monoamine metabolites, 2ā€“250 nM for amino acids, 0.5 nM for acetylcholine, 2 nM for histamine, and 25 nM for adenosine at sample volume of 5 Ī¼L. Relative standard deviation for repeated analysis at concentrations expected in vivo averaged 7% (<i>n</i> = 3). Commercially available <sup>13</sup>C benzoyl chloride was used to generate isotope-labeled internal standards for improved quantification. To demonstrate utility of the method for study of small brain regions, the GABA<sub>A</sub> receptor antagonist bicuculline (50 Ī¼M) was infused into a rat ventral tegmental area while recording neurotransmitter concentration locally and in nucleus accumbens, revealing complex GABAergic control over mesolimbic processes. To demonstrate high temporal resolution monitoring, samples were collected every 60 s while neostigmine, an acetylcholine esterase inhibitor, was infused into the medial prefrontal cortex. This experiment revealed selective positive control of acetylcholine over cortical glutamate

    Reducing Adsorption To Improve Recovery and in Vivo Detection of Neuropeptides by Microdialysis with LC-MS

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    Neuropeptides are an important class of neurochemicals; however, measuring their concentration in vivo by using microdialysis sampling is challenging due to their low concentration and the small samples generated. Capillary liquid chromatography with mass spectrometry (cLC-MS) can yield attomole limits of detection (LOD); however, low recovery and loss of sample to adsorptive surfaces can still hinder detection of neuropeptides. We have evaluated recovery during sampling and transfer to the cLC column for a selection of 10 neuropeptides. Adding acetonitrile to sample eliminated carryover and improved LOD by 1.4- to 60-fold. The amount of acetonitrile required was found to have an optimal value that correlated with peptide molecular weight and retention time on a reversed phase LC column. Treating AN69 dialysis membrane, which bears negative charge due to incorporated sulfonate groups, with polyethylenimine (PEI) improved recovery by 1.2- to 80-fold. The effect appeared to be due to reducing electrostatic interaction between peptides and the microdialysis probe because modification increased recovery only for peptides that carried net positive charge. The combined effects improved LOD of the entire method by 1.3- to 800-fold for the different peptides. We conclude that peptides with both charged and hydrophobic regions require combined strategies to prevent adsorption and yield the best possible detection. The method was demonstrated by determining orexin A, orexin B, and a novel isoform of rat Ī²-endorphin in the arcuate nucleus. Dialysate concentrations were below 10 pM for these peptides. A standard addition study on dialysates revealed that while some peptides can be accurately quantified, some are affected by the matrix

    AICAR effect on HMGR and Central Carbon metabolism.

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    <p>(A) AMPK effect on mevalonate pathway. Cells were incubated with/without AICAR for 1 h, followed by stimulation with 12C glucose for different time points, resulting in the illustrated levels of (B) HMG-CoA, (C) farnesyl pyrophosphate and (D) ratio of farnesyl pyrophophate/ HMG-CoA. Cells were incubated with/without AICAR for 1 h, followed by stimulation with U-<sup>13</sup>C glucose for different time points, resulting in the illustrated levels of different isotopomers of (E) fructose bisphosphate and (F) Citrate. Studentā€™s t-test was performed on all time points comparing the control and AICAR treated samples, with n = 3 or 4 for each replicate.</p

    Effect of AICAR on Ceramides and glycerolipids synthesis pathway.

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    <p>(A) levels of DAG (34:1) isotopomers after stimulation with U-<sup>13</sup>C glucose for 65 minutes. (B) ratio of +3 labeled DAG/unlabeled DAG (C) Glycerolipid and Kennedy pathway for PE synthesis: ECT-ethanolamine phosphate cytidylyltransferase, EPT-ethanolamine phosphotransferase, GPAT-glycerol-3-phosphate O-acyltransferase, (D) CDP-ethanolamine levels after incubation of cells at 2 mM glucose for 60 minutes +/- AICAR +/- phospholipase C (PLC) inhibitor (U-73122) 20 Ī¼M or +/- phospholipase D inhibitor Cay10593 (60 Ī¼M). (E) Ceramide levels after incubation of cells with 50 Ī¼M palmitic acid +/- AICAR 250 Ī¼M for 1 h before stimulation with U-<sup>13</sup>C glucose for different time points. Studentā€™s t-test was performed with n = 3 or 4 for each replicate.</p

    PKCĪ² Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux

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    Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCĪ²) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCĪ² inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum <i>in vitro</i>. In this study, we utilized <i>in vivo</i> microdialysis in live, behaving rats to assess the effect of the PKCĪ² inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 Ī¼M enzastaurin or 1 Ī¼M ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCĪ² inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCĪ² inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCĪ² inhibitors to reduce the effects of amphetamine
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