1. Peptide Serial Dilution
<ol>
	<li>Prepare assay buffer: Hanks buffered salt solution (HBSS) supplemented with 25 mM HEPES, 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), pH 7.4.</li>
	<li>Use a bulk reagent dispenser to systematically add 5 &#181;l of assay buffer to each well of five 384-well low volume assay plates.
	<ol>
		<li>Use internal software to create a dispensing program for 5 &#181;l volume addition to every well of a 384-well plate as per manufacturer&#39;s instructions.</li>
		<li>Immerse dispensing cassette tubing in assay buffer and prime fluid.</li>
		<li>Place 384-well low volume assay plate on plate carrier.</li>
		<li>Press start.</li>
	</ol>
	</li>
	<li>Dilute all peptide samples, regardless of storage vehicle, into assay buffer to produce a 100x peptide stock. 
	NOTE: Peptides requiring screening may be provided in Phosphate-buffered saline (PBS) or Dimethyl sulfoxide (DMSO) as is deemed appropriate (e.g., DMSO will have a deleterious effect on peptides with secondary modifications such as PEGylation).</li>
	<li>Dispense 25 &#181;l 100x peptide stocks into columns 1-5 of an acoustically qualified 384-well polypropylene microplate. This plate is designated &#39;source plate A&#39;. Ensure that source plates, but not destination plates, are flat bottomed and conform to specific acoustic tolerances as defined by the manufacturer of acoustic instruments.</li>
	<li>Dispense 25 &#181;l 100x reference control into wells A23 and A24 of source plate A.</li>
	<li>Dispense 10 &#181;l assay buffer into columns 11-15 and 30 &#181;l assay buffer into columns 21-22 of source plate A.</li>
	<li>Dispense 10 &#181;l assay buffer into columns 6-10 and columns 16-20 of a second acoustically qualified 384-well polypropylene microplate. This plate is designated &#39;source plate B&#39;.</li>
	<li>Centrifuge source plates A and B at 300 x g for 1 min. Include an appropriate balance plate.</li>
	<li>Use acoustic fluid dispensers integrated into an automated robotic system to prepare three sequential 1:100 intermediate dilutions (in assay buffer) of the 100x peptide stocks from column 1 in source A. 
	NOTE: Programming requires plate reformatting and dose response software (as provided by the manufacturer of the acoustic fluid dispenser) to allow three acoustic transfers between source A and source B (see Figure 1 for plate layouts). Step 1.9 details specifically the use of fluid dispensers used in this laboratory under automated robotic control (see Materials Table):
	<ol>
		<li>Load source plates and assay plates into plate hotel section of robotics.</li>
		<li>Open automated robotic software and load plate reformatting and dose response program expansion protocols. Click &#39;Run&#39;. 
		NOTE: Automation instructs an acoustic fluid dispenser to transfer 250 nl from columns 1-5 of source plate A into columns 6-10 of source plate B, and a second acoustic fluid dispenser capable of handling larger fluid volumes to backfill with 15 &#181;l assay buffer to mix. Automation then transfers source plate B to integrated centrifuge and centrifuges at 300 x g for 1 min (an appropriate balance plate is included for all centrifugation steps). Source plate B is returned to the acoustic fluid dispenser for the transfer of 250 nl from columns 6-10 of source plate B into columns 11-15 of source plate A and backfill with 15 &#181;l assay buffer to mix. Automation transfers source plate A to integrated centrifuge and centrifuges at 300 x g for 1 min. Source plate A is then returned to the acoustic fluid dispenser for the transfer of 250 nl from columns 11-15 of source plate A into columns 16-20 of source plate B and backfill with 15 &#181;l assay buffer to mix. Automation then transfers source plate B to integrated centrifuge and centrifuges at 300 x g for 1 min. 
		NOTE: Finally the dose response protocol directs acoustic dispensing to transfer the required volume from each of the 4 serially diluted source plate wells (in both source plate A and source plate B) to construct a full 11-point curve in duplicate in assay plates (pre-filled with 5 &#181;l assay buffer in step 1.2 above). Automation transfers assay plate to integrated centrifuge and centrifuges at 300 x g for 1 min.</li>
	</ol>
	</li>
</ol>
2. Cell Preparation
<ol>
	<li>Thaw cryopreserved Chinese hamster ovary (CHO) cells expressing the target mouse GLP-1 receptor rapidly in a 37 &#186;C water bath and resuspend in 20 ml assay buffer.</li>
	<li>Centrifuge cell suspension for 5 min at 200 x g at room temperature (RT). Include an appropriate balance.</li>
	<li>Discard supernatant and resuspend cell pellet in 10 ml assay buffer.</li>
	<li>Dilute cell stock 1:1 in Trypan blue and determine viable cell density using an automated cell counter. Resuspend cells in assay buffer at 1.6 x 106 cells per ml (equivalent to 8,000 cells per well of assay plates).</li>
	<li>Use a bulk reagent dispenser to add 5 &#181;l of cell suspension to each well of the assay plates (containing serially diluted peptides in 5 &#181;l assay buffer) and incubate at RT for 30 min.</li>
</ol>
3. HTRF cAMP Detection Assay
<ol>
	<li>Bring HTRF cAMP assay kit to RT for 30 min prior to use.</li>
	<li>Prepare each HTRF reagent (cryptate and d2) separately at 1:20 dilution in lysis buffer (proprietary formulation, provided by manufacturer of cAMP detection assay). 
	NOTE: CAUTION: Lysis buffer contains potassium fluoride (KF) which is toxic and a teratogen13. For disposal, assay plates containing KF should be sealed and incinerated, and any liquid waste must be diluted to &#60;1 mmol/L with water prior to disposal down the sink.</li>
	<li>Use a bulk reagent dispenser to add 5 &#181;l cryptate reagent to all wells of the assay plates.</li>
	<li>Use a bulk reagent dispenser to add 5 &#181;l d2 reagent to columns 1-22 of the assay plates.</li>
	<li>Immediately manually pipette 5 &#181;l lysis buffer into wells A23-D24 of the assay plates to give non-specific binding (NSB) control wells, and 5 &#181;l of d2 reagent into wells E23-P24.</li>
	<li>Centrifuge the assay plates for 1 min at 200 x g at RT to mix the wells.</li>
	<li>Cover the assay plates to minimize evaporation and photo-bleaching and incubate at RT for 1 hr.</li>
	<li>Measure fluorescence resonance energy transfer (FRET) signal using excitation at 320 nm and emission at 620 nm and 665 nm using a plate reader.</li>
</ol>
4. Data Analysis
<ol>
	<li>Use mean NSB to subtract background from all wells.</li>
	<li>Calculate %Delta F from the 665 nm/620 nm ratio as follows: 
	Ratio = (A665nm/A620nm) x 104 
	Delta F = ((Sample Ratio - RatioNSB)/RatioNSB)) x 100 
	where RatioNSB = wells with no d2 reagent.</li>
	<li>Calculate % activation between unstimulated cells and cells stimulated with maximum GLP-1 ligand as follows: 
	% activation = (%Delta Fsample - %DeltaFunstimulated cells)/(%DeltaFGLP-1 stimulated cells - %DeltaF unstimulated cells) x 100</li>
	<li>Analyze concentration-response curves via 4-parameter logistical analysis, and graph as % activation as defined by reference control1.</li>
</ol>

<ol>
	<li>Naylor, J., Rossi, A., Hornigold, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+Dispensing+Preserves+the+Potency+of+Therapeutic+Peptides+throughout+the+Entire+Drug+Discovery+Workflow.">Acoustic Dispensing Preserves the Potency of Therapeutic Peptides throughout the Entire Drug Discovery Workflow.</a> J.Lab.Autom. 21 (1), 90-96 (2016).</li><li>Campbell, J. E., Drucker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology,+physiology,+and+mechanisms+of+incretin+hormone+action.">Pharmacology, physiology, and mechanisms of incretin hormone action.</a> Cell.Metab. 17 (6), 819-837 (2013).</li><li>Hui, H., Farilla, L., Merkel, P., Perfetti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+short+half-life+of+glucagon-like+peptide-1+in+plasma+does+not+reflect+its+long-lasting+beneficial+effects.">The short half-life of glucagon-like peptide-1 in plasma does not reflect its long-lasting beneficial effects.</a> Eur.J.Endocrinol. 146 (6), 863-869 (2002).</li><li>Harris, D., Olechno, J., Datwani, S., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient,+contact-free+volume+transfers+minimize+compound+loss+in+dose-response+experiments.">Gradient, contact-free volume transfers minimize compound loss in dose-response experiments.</a> J.Biomol.Screen. 15 (1), 86-94 (2010).</li><li>Ekins, S., Olechno, J., Williams, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispensing+Processes+Impact+Apparent+Biological+Activity+as+Determined+by+Computational+and+Statistical+Analyses.">Dispensing Processes Impact Apparent Biological Activity as Determined by Computational and Statistical Analyses.</a> PLoS ONE. 8 (5), 62325 (2013).</li><li>Goebel-Stengel, M., Stengel, A., Tache, Y., Reeve, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+using+the+optimal+plasticware+and+glassware+in+studies+involving+peptides.">The importance of using the optimal plasticware and glassware in studies involving peptides.</a> Anal.Biochem. 414 (1), 38-46 (2011).</li><li>McDonald, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+Contaminants+Leach+from+Disposable+Laboratory+Plasticware.">Bioactive Contaminants Leach from Disposable Laboratory Plasticware.</a> Science. 322 (5903), 917 (2008).</li><li>Belaiche, C., Holt, A., Saada, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonylphenol+ethoxylate+plastic+additives+inhibit+mitochondrial+respiratory+chain+complex+I.">Nonylphenol ethoxylate plastic additives inhibit mitochondrial respiratory chain complex I.</a> Clin Chem. 55 (10), 1883-1884 (2009).</li><li>Sackmann, E. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achieving+accurate+compound+concentration+in+cell-based+screening:+validation+of+acoustic+droplet+ejection+technology.">Achieving accurate compound concentration in cell-based screening: validation of acoustic droplet ejection technology.</a> J.Biomol.Screen. 14 (5), 452-459 (2009).</li><li>Turmel, M., Itkin, Z., Liu, D., Nie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Innovative+Way+to+Create+Assay+Ready+Plates+for+Concentration+Response+Testing+Using+Acoustic+Technology.">An Innovative Way to Create Assay Ready Plates for Concentration Response Testing Using Acoustic Technology.</a> J.Lab.Autom. 15 (4), 297-305 (2010).</li><li>Butler, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+site-specific+retargeting+jump-in+platform+cell+line+to+support+biologic+drug+discovery.">Use of the site-specific retargeting jump-in platform cell line to support biologic drug discovery.</a> J.Biomol.Screen. 20 (4), 528-535 (2015).</li><li>Panchal, S., Verma, R. 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Aided Mol.Des. 29 (12), 1073-1086 (2015).</li><li>Harris, D., Olechno, J., Datwani, S., Ellson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient,+Contact-Free+Volume+Transfers+Minimize+Compound+Loss+in+Dose-Response+Experiments.">Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments.</a> J.Biomol. Screen. 15 (1), 86-94 (2010).</li><li>Chan, G. K. Y., Wilson, S., Schmidt, S., Moffat, J. 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The authors have nothing to disclose.

This protocol describes the successful application of automated acoustic dispensing to serially dilute peptide samples over a concentration range of 3 x 106 requiring less than 1 &#181;l of sample. The major advantage of this method is to increase data quality through minimizing peptide adsorption to plasticware via reduced exposure of samples to experimental containers and plasticware (such as pipette tips) that are normally required for reagent transfer and mixing. While acoustic dispensing does not complete...

The GLP-1R is an established drug target in the treatment of type 2 diabetes2. The native peptide agonist for this receptor, GLP-1, has an in vivo half-life of 2-3 min3. The binding of GLP-1 to its G protein coupled target receptor results in the downstream production of the second messenger cAMP through native G protein coupling to the activation of adenylyl cyclase. Measurement of the accumulated cAMP provides a robust assay to monitor receptor activation and to screen for active GLP-1 analogues with preferred physicochemical properties. Such an assay requires the serial dilution of test samples to construct concentration-response curves, and this is particularly complicated when handing peptide samples. Potential errors from tip-based serial dilution preparation have been described previously1,4,5. Peptides will adsorb to plasticware, resulting in unreliable potency estimations. Peptide loss can be minimized through the inclusion of bovine serum albumin (BSA) in buffers and the use of siliconized plasticware, yet protein binding remains unpredictable. In particular, the variation in binding of GLP-1 to experimental containers has been described6. There is a further complication in that stabilization agents used in laboratory plasticware can leach from tips and microtiter plates into aqueous assay buffers and interfere with protein function7, 8. Therefore, methods to reduce exposure to plasticware are necessary to increase the accuracy of measurements.
Acoustic liquid dispensers focus a high-frequency acoustic signal onto the surface of a fluid sample, resulting in the ejection of precise nanoliter droplets into an adjacent assay plate9. The use of acoustic ejection is standard in the pharmaceutical industry for the preparation and screening of large synthetic compound libraries, and the technology has been well validated for small molecules10. To our knowledge, we are the first group to describe acoustic dispensing for the preparation of recombinant and synthetic peptides and we have previously reported the improved accuracy compared to conventional tip-based methods1.
This article describes the integration of the preparation of peptide serial and direct dilutions by non-contact acoustic transfer onto a fully automated plate handling robotics system. A number of methods encompassing acoustic transfer of samples have been described previously11. We utilize a two-step method to prepare intermediate stock concentrations and to serially dilute peptide analogues for the generation of the full dose-response curve. The prepared peptides are incubated with cells expressing the target mouse GLP-1R, and we use a commercially available homogenous time-resolved fluorescence (HTRF) assay to measure cAMP accumulation within these cells as a readout of peptide agonist activity. The assay is robust and amenable to a high-throughput 384-well format and routinely applied to both assay development and drug screening projects12.

<table><tbody><tr><td>Hanks&amp;rsquo; Balanced Salt solution</td><td>Sigma-Aldrich</td><td>H8264</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I7018</td><td>Prepared as a 0.5 M stock in DMSO</td></tr><tr><td>GLP-1 (7-36) amide</td><td>Bachem</td><td>H-6795</td><td>Prepared as a 1 mg/ml stock in PBS, referred to as &#039;100x reference control&#039;</td></tr><tr><td>Test peptides</td><td>Produced in-house at MedImmune</td><td></td><td>Supplied at various concentrations in DMSO or PBS as appropriate</td></tr><tr><td>100x peptide stock</td><td>Produced in-house at MedImmune</td><td></td><td>Test peptide diluted into assay buffer to 100x final required concentration</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250-061</td><td></td></tr><tr><td>Cedex XS Cell Analyzer</td><td>Innovatis</td><td></td><td></td></tr><tr><td>Corning 384 well plates, low volume</td><td>Sigma-Aldrich</td><td>4514</td><td></td></tr><tr><td>Echo Qualified 384-Well Polypropylene Microplate</td><td>Labcyte Inc.</td><td>P-05525</td><td></td></tr><tr><td>Echo Qualified Reservoir</td><td>Labcyte Inc.</td><td>ER-0055</td><td></td></tr><tr><td>Echo 550 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 2.5 nl</td></tr><tr><td>Echo 525 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 25 nl</td></tr><tr><td>ACell Benchtop Automation&amp;nbsp;</td><td>HighRes Biosolutions</td><td>MC522</td><td></td></tr><tr><td>Cellario Lab Automation Scheduling software for Life Science Robotics</td><td>HighRes Biosolutions</td><td></td><td></td></tr><tr><td>MultidropCombi Reagent Dispenser</td><td>ThermFisher Scientific</td><td>5840300</td><td>Referred to as &#039;bulk reagent dispenser&#039;</td></tr><tr><td>HTRF cAMP Dynamic 2 kit</td><td>Cisbio Bioassays</td><td>62AM4PEJ</td><td></td></tr><tr><td>EnVision Multilabel Reader</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

We routinely use a two-step method to dilute peptides via acoustic transfer. For the first step, an acoustic dispenser aligned with automation is used to create four stock peptide intermediate dilutions across two source plates (Figure 1a, b). For the second step, we use an acoustic dispenser to further dilute stock dilutions from source plates A and B to create an 11-point concentration range for each test peptide (Figure 1c). Each pepti...

Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

automated-acoustic-dispensing-for-serial-dilution-peptide-agonists

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

7.6K Views.  MedImmune. Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described. 

Video: Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Chemistry

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Bioengineering

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Immunology and Infection

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome ("surfaceome") in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biology

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...