Name: automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays
Uploaded: 2016-11-10T12:30:00
Description: Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

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 disp...

<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. K., 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=Technologies+That+Enable+Accurate+and+Precise+Nano-+to+Milliliter-Scale+Liquid+Dispensing+of+Aqueous+Reagents+Using+Acoustic+Droplet+Ejection.">Technologies That Enable Accurate and Precise Nano- to Milliliter-Scale Liquid Dispensing of Aqueous Reagents Using Acoustic Droplet Ejection.</a> J.Lab.Autom. 21 (1), 166-177 (2016).</li><li>Grant, R. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sodium+fluoride+in+maternal+and+offspring+rats+and+its+amelioration.">Effect of sodium fluoride in maternal and offspring rats and its amelioration.</a> Asian Pac.J.Reprod. 3 (1), 71-76 (2014).</li><li>Hanson, S. M., Ekins, S., Chodera, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+error+in+experimental+assays+using+the+bootstrap+principle:+understanding+discrepancies+between+assays+using+different+dispensing+technologies.">Modeling error in experimental assays using the bootstrap principle: understanding discrepancies between assays using different dispensing technologies.</a> J.Comput. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlocking+the+potential+of+high-throughput+drug+combination+assays+using+acoustic+dispensing.">Unlocking the potential of high-throughput drug combination assays using acoustic dispensing.</a> J.Lab.Autom. 21 (1), 125-132 (2016).</li><li>Roberts, K., 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=Implementation+and+challenges+of+direct+acoustic+dosing+into+cell-based+assays.">Implementation and challenges of direct acoustic dosing into cell-based assays.</a> J.Lab.Autom. 21 (1), 76-89 (2016).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1339">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Hanks&#8217; Balanced Salt solution</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8264</td>
			<td style="width:517px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">HEPES</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Bovine Serum Albumin</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">I7018</td>
			<td>Prepared as a 0.5 M stock in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">GLP-1 (7-36) amide</td>
			<td style="width:235px;">Bachem</td>
			<td>H-6795</td>
			<td>Prepared as a 1 mg/ml stock in PBS, referred to as &#39;100X reference control&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Test peptides</td>
			<td style="width:235px;">Produced in-house at MedImmune</td>
			<td></td>
			<td>Supplied at various concentrations in DMSO or PBS as appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">100X peptide stock</td>
			<td style="width:235px;">Produced in-house at MedImmune</td>
			<td></td>
			<td>Test peptide diluted into assay buffer to 100X final required concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:235px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cedex XS Cell Analyzer</td>
			<td style="width:235px;">Innovatis</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Corning 384 well plates, low volume</td>
			<td style="width:235px;">Sigma-Aldrich</td>
			<td style="width:119px;">4514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo Qualified 384-Well Polypropylene Microplate</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;">P-05525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo Qualified Reservoir</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;">ER-0055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo 550 Liquid Handler</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;"></td>
			<td>Droplet transfer volumes in increments of 2.5 nl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Echo 525 Liquid Handler</td>
			<td style="width:235px;">Labcyte Inc.</td>
			<td style="width:119px;"></td>
			<td>Droplet transfer volumes in increments of 25 nl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">ACell Benchtop Automation&#160;</td>
			<td style="width:235px;">HighRes Biosolutions</td>
			<td style="width:119px;">MC522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Cellario Lab Automation Scheduling software for Life Science Robotics</td>
			<td style="width:235px;">HighRes Biosolutions</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">MultidropCombi Reagent Dispenser</td>
			<td style="width:235px;">ThermFisher Scientific</td>
			<td style="width:119px;">5840300</td>
			<td>Referred to as &#39;bulk reagent dispenser&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">HTRF cAMP Dynamic 2 kit</td>
			<td style="width:235px;">Cisbio Bioassays</td>
			<td style="width:119px;">62AM4PEJ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">EnVision Multilabel Reader</td>
			<td style="width:235px;">PerkinElmer</td>
			<td style="width:119px;"></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. ...

Research

JoVE Journal

Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

In organ transplantation, the function and longevity of the graft critically rely on the success of controlling immunological rejection reactivity against ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...