27 research outputs found
Droplet Electrospray Ionization Mass Spectrometry for High Throughput Screening for Enzyme Inhibitors
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 āMS 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 145ā000
samples to be analyzed in 1 day
Western Blotting Using Microchip Electrophoresis Interfaced to a Protein Capture Membrane
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 11ā155 kDa were
completed in 2 min with 4 Ć 10<sup>4</sup> theoretical plates
at 460 V/cm. Migration time and peak area relative standard deviations
were 3ā6% 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 22ā32 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 (ā¼5 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
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 ā¼0.8. 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 CellāCell Interactions
Microfluidics
have been used to create ābody-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 cellācell 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 190āÆ000 3T3-L1 adipocytes
and a single islet of Langerhans in separate chambers. The chambers
were perfused at 300ā600 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 cellācell interaction
can provide valuable insights into cellular functions
Microfabricated Sampling Probes for in Vivo Monitoring of Neurotransmitters
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 ChromatographyāMass Spectrometry
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)ātandem
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 0.03ā0.2 nM for monoamine neurotransmitters,
0.05ā11 nM for monoamine metabolites, 2ā250 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
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.
<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. Studentās 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.
<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. Studentās t-test was performed with n = 3 or 4 for each replicate.</p
PKCĪ² Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux
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