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>
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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. 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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. 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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 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. ...

The overall goal of this automated protocol for the serial dilution of peptide samples is to minimize peptide absorption to plasticware enabling accurate potency determination for drug discovery. This method can increase the quality and reproducibility of data and the removal of tip-based pipetting also eliminates sample carryover so that errors are not propagated throughout the entire serial dilution. The main advantage of this technique is that it increases data quality by minimizing peptide absorption to plasticware.This is particularly important for driving a structure-activity relationship or lead identification campaign in drug discovery. After preparing the assay buffer and creating a dispensing program for a bulk dispenser according to the text protocol, immerse dispensing cassette tubing in assay buffer and prime with fluid. Place a 384 well low volume assay plate on a plate carrier.Then press start. Dispense 25 microliters of 100 times peptide stocks into columns one through five of an acoustically qualified 384 well flat-bottomed polypropylene microplate to be designated as source plate A.Dispense 25 microliters of 100 times reference control into wells A23 and A24. Then dispense 10 microliters of assay buffer into columns 11 through 15 and 30 microliters of assay buffer into columns 21 through 22.Next, into columns six through 10 and 16 through 20 of a second acoustically qualified 384 well polypropylene microplate designated source plate B, dispense 10 microliters of assay buffer. Centrifuge source plates A and B at 300 times g for one minute. Next, load the source and assay plates into the plate hotel section of the robotics.Then running the robotics software according to the text protocol, use acoustic fluid dispensers integrated into the system to prepare three sequential one in 100 intermediate dilutions. Start with transferring from columns one to five on source plate A to columns six to 10 on source plate B.After preparing the first one in 100 dilution, allow automation to transfer source plate B to an integrated centrifuge and spin at 300 times g for one minute before returning the plate to the acoustic fluid dispenser. Then after transferring the solution from source plate B to source plate A for the second one in 100 dilution, automation will transfer source plate A to the centrifuge and spin before returning the plate to the fluid dispenser.Finally, after automation transfers the solution from plate A to B for the third one in 100 dilution, plate B is again centrifuged. These peptide dilutions in source plate A and source plate B are then used to prepare full concentration response curves in the assay as described in the text protocol. This step is critical as a comparative tip-based methodology causes a dramatic loss in sample potency due to peptide absorption to plasticware.Rapidly thaw cryopreserved CHO cells expressing the target mouse GLP1 receptor in a 37 degrees water bath and use 20 milliliters of assay buffer to resuspend them. Centrifuge the cell suspension at 200 times g and room temperature for five minutes. Discard the supernatant and use 10 milliliters of assay buffer to resuspend the cell pellet.With Trypan Blue, dilute the cell stock one to one and use an automated cell counter to determine the viable cell density. Then use assay buffer to resuspend the cells at a concentration of 1.6 million cells per milliliter. Using a bulk reagent dispenser, add five microliters of cell suspension to each well of the assay plate containing serially diluted peptides and incubate at room temperature for 30 minutes.To carry out the cyclic AMP detection assay, use the lysis buffer to prepare one in 20 dilutions of each homogenous time-resolved fluorescence or HTRF reagent. Use a bulk reagent dispenser to add five microliters of cryptate reagent to all the wells of the assay plates. Then add five microliters of D2 reagent to columns one through 22 of the plate.Next, add five microliters of D2 reagent into wells E23 through P24. Immediately use a manual pipette to add five microliters of lysis buffer to wells A23 through D24 to be used as nonspecific binding control wells. Centrifuge the assay plates at 200 times g and room temperature for one minute to mix the wells.Then cover the plates and incubate for one hour. After the incubation, use a plate reader to measure the fret signal at an excitation of 320 nanometers and an emission of 620 and 665 nanometers. Analyze the data according to the text protocol.The first step in acoustic transfer for the cyclic AMP detection assay is to create four stock peptide intermediate dilutions across two source plates as shown here. For the second step, the acoustic dispenser further dilutes stock solutions from source plates A and B to create an 11-point concentration range for each test peptide in duplicate for 16 different peptides. Assay buffer backfill is performed to ensure a constant volume per well across the assay plate.Shown here is a heat map for an example plate containing 16 peptides in duplicate run through the cyclic AMP accumulation assay. Low values shown in purple represent high cyclic AMP concentration. These concentration response curves illustrate the reproducibility of duplicate points.Sample one represents a positive control for the assay. Samples two, three, and four were inactive and are represented by the flat lines on the graph. Samples five through 16 represent active test peptides.The method presented here allows for the generation of full concentration response curves across a broad potency range. Once mastered, this technique can be completed in three hours, but this will depend on the number of samples requiring preparation. While attempting this procedure, it is important to remember that peptides are biological samples and should be handled as such.In order to maintain their integrity, repeat freeze-thaw cycles should be avoided. This technique will pave the way for researchers in the field of peptide drug discovery and allow for the accurate potency predictions needed to fuel structure-activity relationships. After watching this video, you should have a good understanding of automated acoustic dispensing for the serial dilution of peptides and this will minimize peptide loss by absorption and increase the accuracy of your measurements.Don't forget that working with biologically active peptides with picomolar potencies can be extremely hazardous and precautions such as personal protective equipment should always be used.

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Biochemistry

7.5K ビュー.  MedImmune. The overall goal of this automated protocol for the serial dilution of peptide samples is to minimize peptide absorption to plasticware enabling accurate potency determination for drug discovery. This method can increase the quality and reproducibility of data and the removal of tip-based pipetting also eliminates sample carryover so that errors are not propagated throughout the entire serial dilution. The main advantage of this technique is that it increases data quality by minimizing peptid...