Name: stepwise dosing protocol for increased throughput in label-free impedance-based gpcr assays
Uploaded: 2020-02-21T13:00:00
Description: Watch this Scientific Journal Video about Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays at JoVE.com

We thank Barbara Goricnick and Nadja Hinterreiter for their help with cell culturing and preparation of experimental solutions. The authors gratefully acknowledge financial support by the Research Training Group 1910 &#34;Medicinal chemistry of selective GPCR ligands&#34; funded by the German Research Foundation (DFG) under grant number 222125149. JAS is particularly grateful for a scholarship granted by the Bavarian Gender Equality Program.

1. Cell seeding on electrode arrays
NOTE: Selection of electrode layout is a trade-off between sensitivity and number of cells under study. The smaller the electrode, the more sensitive is the measurement but the smaller is the number of cells under study. For cells showing strong impedance fluctuations over time under baseline conditions, bigger or interdigitated electrodes are preferable.
<ol>
	<li>Pre-warm all solutions needed for standard cell passaging and seeding in a 37 &#176;C water ...

<ol>
	<li>Rosenbaum, D. M., Rasmussen, S. G., Kobilka, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+function+of+G-protein-coupled+receptors.">The structure and function of G-protein-coupled receptors.</a> Nature. 459, 356-363 (2009).</li><li>Sriram, K., Insel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein-Coupled+Receptors+as+Targets+for+Approved+Drugs:+How+Many+Targets+and+How+Many+Drugs.">G Protein-Coupled Receptors as Targets for Approved Drugs: How Many Targets and How Many Drugs.</a> Molecular Pharmacology. 93, 251-258 (2018).</li><li>Kaitin, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+drug+development+process:+The+new+face+of+innovation.">Deconstructing the drug development process: The new face of innovation.</a> Clinical Pharmacoly and Therapeutics. 87, 6 (2010).</li><li>Kenakin, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+assays+as+portals+to+seven-transmembrane+receptor-based+drug+discovery.">Cellular assays as portals to seven-transmembrane receptor-based drug discovery.</a> Nature reviews. Drug discovery. 8, 617-626 (2009).</li><li>Zhang, R., Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+GPCR+drug+discovery.">Tools for GPCR drug discovery.</a> Acta Pharmacologica Sinica. 33, 372-384 (2012).</li><li>Scott, C. W., Peters, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+whole-cell+assays:+expanding+the+scope+of+GPCR+screening.">Label-free whole-cell assays: expanding the scope of GPCR screening.</a> Drug Discovery Today. 15, 704-716 (2010).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proceedings of the National Academy of Sciences of the United States of America. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morphological+biosensor+for+mammalian+cells.">A morphological biosensor for mammalian cells.</a> Nature. 366, 591-592 (1993).</li><li>Fang, Y., Ferrie, A. M., Fontaine, N. H., Mauro, J., Balakrishnan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resonant+waveguide+grating+biosensor+for+living+cell+sensing.">Resonant waveguide grating biosensor for living cell sensing.</a> Biophysical Journal. 91, 16 (2006).</li><li>Stolwijk, J. A., Matrougui, K., Renken, C. W., Trebak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+analysis+of+GPCR-mediated+changes+in+endothelial+barrier+function:+overview+and+fundamental+considerations+for+stable+and+reproducible+measurements.">Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.</a> Pflugers Archiv : European Journal of Physiology. 467, 2193-2218 (2015).</li><li>Lieb, S., 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=Label-free+versus+conventional+cellular+assays:+Functional+investigations+on+the+human+histamine+H1+receptor.">Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.</a> Pharmacological Research. 114, 13-26 (2016).</li><li>Stolwijk, J. A., 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=Increasing+the+throughput+of+label-free+cell+assays+to+study+the+activation+of+G-protein-coupled+receptors+by+using+a+serial+agonist+exposure+protocol.">Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.</a> Integrative Biology. 11, 10 (2019).</li></ol>

This protocol describes a method for label-free impedance measurements to determine the dose-response relationship of agonist-induced GPCR activation in absence or presence of specific antagonists for the same receptor. The proof of concept of this method was presented in a recent publication12. To our knowledge it is the first study describing the establishment of a full dose-response curve of agonist-mediated GPCR activation using a single cell layer in vitro. The approach inevitably requires th...

This report presents a detailed description of a serial addition protocol developed for the quantification of ligand-induced G protein-coupled receptor (GPCR) activation in adherently grown cells by label-free impedance measurements. G protein-coupled receptors (GPCRs) are involved in a multitude of physiological functions and human diseases1. Because of this and their good accessibility at the cell surface, GPCRs are one of the most important drug targets. This assessment is reflected in an estimated number of ~700 approved drugs targeting GPCRs, equivalent to a ~35% share on all marketed drugs2.
The development of new drugs comprises two central processes: (i) the identification and functional characterization of biological target molecules, and (ii) the discovery of new lead substances and their development into administrable drugs. In both processes, efficient methods are required to quantitatively evaluate drug-target interactions and the subsequent biological downstream response. Different stages of the pre-clinical drug development process make use of different methods of analysis ranging from biomolecular interaction studies between drug and target, over functional studies on cells in culture, to experiments on excised organ material or whole animals. Both, physiological significance and biological complexity increase from the former to the latter3. Although it is the overall goal to minimize animal experiments, pharmacological studies using isolated organs from laboratory animals or even whole animals are regarded inevitable to comprehensively characterize new drug candidates. In terms of analytical readout, organ pharmacology studies provide a distal, integrative &#34;holistic&#34; functional response of highest physiological relevance. A drawback of such experiments is that they are not compatible with high throughput screening for technical as well as ethical reasons and have been largely substituted by studies based on in vitro cell culture4.
Methods to quantify GPCR activation in cell cultures include different label-based chemical assays, which specifically detect second messengers, phosphorylation state of downstream signaling proteins, transcriptional activation via certain transcription factors or ligand-induced intracellular receptor trafficking4,5. A drawback of such label-based assays is the necessity to label the cells with potentially harmful dyes or radioactive markers. This often requires running the assay as an endpoint-determination for an exposure time that has to be specified a priori. Using label-based endpoint assays suffers from this very limited and biased timing of the experiment and the risk that chemical labels and probes may interfere with the regular cell physiology &#8211; potentially unnoticed by the experimenter.
In recent years, label-free assays for monitoring GPCR activation have emerged, like impedance-based techniques or optical methods applying resonant waveguide gratings5,6. Labelling of the cells is experimentally not required with these approaches. As these physical readouts operate on low amplitude signals, such methods are considered non-invasive, they allow for continuous cell monitoring potentially in real time and the observation time is only limited by the cell culture not by the readout. Similar to readouts from whole organs, label-free approaches commonly report on holistic cell responses, far downstream of receptor activation when integration along the entire signaling network leads to time-dependent but rather unspecific changes in cell morphology or mass redistribution. Whereas impedance-based assays measure the dielectric signature of changes in cell shape7,8, measurements using resonant waveguide gratings are sensitive to changes in refractive index at the cell-substrate interface arising from dynamic mass redistribution (DMR)9. The integrative character makes label-free methods extremely sensitive to receptor-mediated events regardless of the type(s) of G protein (Gq, Gi/0, Gs, G12/13) or &#946;-arrestin involved in the signaling cascade6 and well-suited for endogenous expression levels of the receptor.
In a standard label-free impedance-based assay the cells are adherently grown in multi-well plates with coplanar gold-film electrodes deposited on the bottom of each well10. These electrode arrays are connected to an impedance analyzer and the cell responses to an experimental stimulus are recorded from individual wells by time-resolved impedance readings. In a typical GPCR assay a ligand is added in individually different concentrations to each individual well. The ligand-induced changes in the impedance time course are then analyzed with respect to characteristic curve features such as maximum signal change, area under the curve, signal change within a given time interval or slope of the curve at a specified time point, in order to quantify the ligand&#39;s potency and efficacy11.
The cost of the electrode arrays may limit the application of this technique in high throughput screening (HTS) campaigns. Moreover, with increasing numbers of samples to be followed in parallel, the number of individual measurements increases and thereby reduces the available time resolution for each well gradually - even for state of the art multichannel recordings. Under such conditions fast and transient cell responses may escape the measurement. In addition, the conventional one well &#8211; one concentration approach imposes a significant time and cost factor on perfused organ-on-chip or body-on-chip developments with respect to their suitability in ligand-GPCR interaction analysis.
For this reason, we developed a stepwise dosing protocol that enables recording of full dose-response curves of ligand-induced GPCR activation in cultured cell monolayers by continuously monitoring the impedance of a single well while the agonist concentration is increased stepwise. The serial agonist addition protocol significantly increases throughput per well from one concentration to 10 or more concentrations, as shown on the current example of human U-373 MG cells, which endogenously express the histamine 1 receptor (H1R). Thus, the method has the potential to significantly improve throughput in label-free dose-response studies, while the time resolution is retained at the instrumental maximum.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>B&#252;rker counting chamber</td>
			<td>Marienfeld (Lauda-K&#246;nigshofen, Germany)</td>
			<td>640210</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture flasks 25 cm2</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>690175</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell incubator (Heraeus Function Line BB15)</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (Heraeus 1S-R)</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>Diphenhydramine hydrochloride</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>D3630</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#39;s Minimum Essential Medium with 4.5 g/L D-Glucose and 2.2 g/L NaHCO3</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>Impedance Instrument (ECIS Z&#952;)</td>
			<td>Applied BioPhysics Inc. (Troy, NY, USA)</td>
			<td>\</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well electrode Arrays (8W1E PET)</td>
			<td>Applied BioPhysics Inc. (Troy, NY, USA)</td>
			<td>\</td>
			<td>PET base with 0.049 mm2 working electrode and ~50 mm2 counter electrode (gold)</td>
		</tr>
		<tr>
			<td>96-well electrode arrays (96W1E+ PET)</td>
			<td>Applied BioPhysics Inc. (Troy, NY, USA)</td>
			<td>\</td>
			<td>PET base with two electrodes (gold) with 0.256 mm2 total electrode area</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Biochrom (Berlin, Germany)</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Carl Roth (Karlsruhe, Germany)</td>
			<td>4017.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood (Herasafe, KS 12)</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>51022515</td>
			<td>class II safety cabinet</td>
		</tr>
		<tr>
			<td>Leibovitz&#39; L-15 medium</td>
			<td>Thermo Scientific (Darmstadt, Germany)</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette large (100 - 1000 &#181;L)</td>
			<td>Brandt (Wertheim, Germany)</td>
			<td>704780</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette large (20 - 200 &#181;L)</td>
			<td>Brandt (Wertheim, Germany)</td>
			<td>704778</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (phase contrast, Nikon Diaphot)</td>
			<td>Nikon (D&#252;sseldorf, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, serological</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>607 180</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (accu-jet pro)</td>
			<td>Brandt (Wertheim, Germany)</td>
			<td>26300</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma Aldrich (Taufkirchen, Germany)</td>
			<td>T4174</td>
			<td>in PBS with 1 mM EDTA</td>
		</tr>
		<tr>
			<td>Tube, 15 mL</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>188 271</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube, 50 mL</td>
			<td>Greiner bio-one (Frickenhausen, Germany)</td>
			<td>210 261</td>
			<td></td>
		</tr>
		<tr>
			<td>U-373 MG cells</td>
			<td>ATCC (Rockville, MD, USA)</td>
			<td>ATCC HTB-17</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath (TW21)</td>
			<td>Julabo (Seelbach, Germany)</td>
			<td>\</td>
			<td></td>
		</tr>
	</tbody>
</table>

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).

A typical scheme for preparing the various agonist solutions is shown for an experiment using 8-well electrode arrays with histamine as agonist in Tables 1-4. Table 1 and Table 2 present volumes and concentrations for an experiment using addition mode 1 (cf., Figure 1), while Table 3 and Table 4 present volumes and concentrations for an experiment following addition mode 2 (...

Watch this Scientific Journal Video about Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays at JoVE.com

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

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 ...

This protocol enables the recording of a complete functional concentration response relationship of GPCR agonist from one cell population and provides detailed agonist characterization even for low cell sample numbers. The main advantage of serial dosing is the increase in throughput since a single well is sufficient for a full concentration response analysis. This technique is well suited to study single transduction in rare and complex biological models, like organ-on-chip formats in closed microfluidic containers.The protocol is not difficult to perform, provided the appropriate equipment is available. Preparing the various GPCR ligand solutions at the right concentration is the key to success. Michael Skiba, a graduate student, and Anne Mildner, an undergraduate student, from our laboratory will demonstrate the procedure.For cell seeding onto the electrode arrays, add the appropriate concentration of cells to the wells of each electrode array, and allow the cells to settle homogenously onto the well bottoms for 10 to 15 minutes at room temperature. After at least 36 hours in a standard cell culture incubator at 37 degrees Celsius and 5%carbon dioxide, inspect the cell layers by phase-contrast microscopy to ensure a complete coverage of each electrode with cells. Then replace the cell culture medium in each well with the appropriate volume of pre-warmed, serum-free medium.To start an impedance recording, place the electrode array into the connecting array holder of the impedance analyzer, and confirm a proper low impedance contact between the electrodes and the impedance analyzer. Select the electrode type and/or multi-well format in the user interface of the software. If single and multiple frequency data acquisition modes are available and the number of wells to be analyzed is low or the time resolution is not critical, select multiple frequency recordings.To ensure a maximum time resolution, select a single monitoring frequency. Then start acquisition of the time course data, and select the data folder for where to save the data. The impedance readings will be recorded at the specified number of frequencies for every well To initiate the serial addition protocol in the agonist mode for an eight-well array, add 30 microliters of the lowest concentration of the agonist of interest to the cells.Then let the cells respond and equilibrate for the predefined period of time before adding 30 microliters of the next highest concentration until each serial dilution of the agonist has been added. To analyze the data, subtract the impedance of the last data point before the first addition of agonist solution, and set the time of addition zero to normalize the impedance values. Plot the time course of normalized impedance, and plot the individual time courses to identify the maxima in impedance after each addition step.Compose a data sheet with these values, and plot the values of maximum impedance change as a function of the agonist concentration. Then use a data-fitting routine to determine the half-maximal effective concentrations and maximum response using the four-parameter logistic model. In this representative experiment, 10 solutions with increasing histamine concentrations were sequentially added to the cells every 15 minutes, and the change in impedance was measured at each time point.The impedance changes could then be plotted as a function of histamine concentration and the transfer function of a four-parameter logistic model fitted to the experimental data points from seven wells. To account for possible histamine receptor downregulation, cell desensitization, or overstimulation, the data range for the analysis can be reduced, as shown for this single serial dosing experiment. Three-parameter optimization can then be applied to the data, providing a half-maximal effective concentration of 0.75 plus or minus 0.12 micromolar for this analysis.The addition of the histamine receptor antagonist diphenhydramine 20 minutes before the first histamine results in a delayed increase of impedance within the serial dosing scheme, corresponding to the need for a higher agonist to elicit a cell response. In the dose response relationship, the effect of the antagonist is expressed as a rightward shift of the curves, corresponding to an increase in half-maximal effective concentration, provided the antagonist is a competitive ligand relative to the agonist. Successful application of the serial dosing scheme is technically easy but requires thoughtful planning, as it is challenging to perform all of the steps with the correct timing.Serial dosing has paved the way for performing concentration response studies with other readout systems that are limited with respect to the number of samples monitored in parallel. The particular advantages of serial dosing allow the study of receptor-mediated signal transduction with completely new readouts that are sensitive to other phenotypic changes, like cytomechanics. Please wear gloves and goggles when preparing the stock solutions of the receptor agonist, as they may be hazardous when inhaled or swallowed.

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Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

Monitoring GPCR-&amp;#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Parallel Interrogation of &amp;#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...