SR and LDU would like to acknowledge Alberta Innovates Strategic Research Project, NRC, and NSERC-Discovery grant for this project.

1. Design and development of peptide library
<ol>
	<li>To identify the ligands of the mast cell MRGPRX2 receptor based on a known ligand i.e., PAMP-1213, follow the steps below.
	<ol>
		<li>Generate N-truncated peptide library by truncating the N-terminal amino acid residues of the ligand, in succession, by solid-phase peptide synthesis (SPPS).</li>
		<li>Generate C-truncated peptide library by truncating the C-terminal amino acid residues of the known ligand, in succession, by SPPS.</li>
		<li>Based on the results of 1.1.1 and 1.1.2, generate an N+C-truncated peptide library using SPPS by truncating the desired residues from the N and C-terminal, respectively.</li>
		<li>Use solid-phase peptide synthesis to synthesize the peptides13.</li>
		<li>Modify the N-terminal to acetyl (Ac) group and C-terminal to amide group.</li>
		<li>Characterize the peptide for its purity using high-pressure liquid chromatography (HPLC) and for the mass using a mass spectrophotometer.</li>
	</ol>
	</li>
	<li>To study the significance of specific amino acids within the parent PAMP-12 molecule, follow the steps below.
	<ol>
		<li>Generate an alanine scanning peptide library by replacing the respective amino acid residues in the peptide molecule with alanine, one at a time using SPPS. Modify the N-terminal to acetyl (Ac) group and C-terminal to amide group.</li>
		<li>Characterize the peptide for its purity using high-pressure liquid chromatography (HPLC) and for the mass using a mass spectrophotometer. 
		NOTE: Ensure that the synthesized peptides are of high purity. Characterize the peptides using mass spectroscopy and HPLC.</li>
	</ol>
	</li>
</ol>
2.&#160;&#160;In vitro cell culture
<ol>
	<li>Culture HEK-X2 and HEK-WT cells by following the steps below.
	<ol>
		<li>Prepare culture medium by supplementing high glucose DMEM with 10% fetal bovine serum (FBS), 2 mM L-Glutamine, 100 U/mL of penicillin and 100 &#181;g/mL of streptomycin.</li>
		<li>Passage the cells in tissue culture (TC) treated T-75 culture flasks and grow in an incubator at 37 &#176;C containing 5% CO2, till they are 75-80% confluent.</li>
		<li>Once 75% confluent, wash the cells and add 2-3 mL of trypsin for 2 - 3 min. Incubate in a 37 &#176;C, 5% CO2 incubator to detach the cells.</li>
		<li>Once the cells have detached, collect the cells in trypsin. Add 6-9 mL of fresh medium.</li>
		<li>Centrifuge the cells at 1620 x g for 3-5 min.</li>
		<li>After centrifugation, discard the supernatant to collect the pellet. Resuspend the cells in a fresh culture medium. Dilute the cells as per the desired concentration. 
		NOTE: HEK cells are fast-growing cells and hence optimize the cell medium supplements. HEK cells are adherent cells; passage them in TC-treated culture flasks to support adhesion.</li>
	</ol>
	</li>
	<li>Prepare a 96 well assay plate for the experiment.
	<ol>
		<li>Add 200 &#181;L of the cell suspension in each well, with a concentration of 200,000 cells/mL, to seed 40,000 cells/well.</li>
		<li>Grow the cells for 24 h in a 37 &#176;C, 5% CO2 incubator. 
		NOTE: Optimize the cell density per well based on the plate size and the cell strain type. Conduct the experiment in triplicates in a black TC-treated 96 well plate with flat transparent bottom.</li>
	</ol>
	</li>
</ol>
3. Fura-2 AM calcium assay
<ol>
	<li>Prepare dye by following the steps below.
	<ol>
		<li>Use Fura-2 AM dye for the experiment.</li>
		<li>Prepare HEPES-Tyrode&#39;s buffer (HTB) buffer containing 25 mM HEPES buffer, 120 mM NaCl, 5 mM KCl, 1 mg/mL glucose, 1 mg/mL bovine serum albumin (BSA) and freshly added 1.8 mM CaCl2 in autoclave sterilized water.</li>
		<li>Add 50 &#181;L of DMSO in 50 &#181;g Fura-2 AM vial to prepare 1 mM stock solution of Fura-2 AM dye. Add 1 &#181;L of 1 mM Fura-2 AM dye per mL of fresh medium to prepare the dye loading medium having 1 &#181;M dye concentration.</li>
		<li>Remove the 96 well plate from the incubator and discard the medium. Replace the medium with a fresh dye loading medium. Add 200 &#181;L of dye loading medium in each well. Incubate the cells for 30-40 min in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>After 40 min of incubation, remove the medium. Wash the cells with the HTB buffer. Add 100 &#181;L of HTB buffer for fluorescence reading. Take the plate for fluorescence reading. 
		NOTE: Optimize the concentration of dye in the dye loading medium. Dye leakage and photobleaching are possible concerns associated with the dye. Add CaCl2 fresh into the HTB buffer to avoid precipitation.</li>
	</ol>
	</li>
</ol>
4. Cell activation and fluorescent reading
NOTE: Fluorescence plate reader with an automated pipetting system allows for the automatic transfer of compounds from a compound source to the assay plate without taking the plate out of the plate reader.
<ol>
	<li>While the cells are being incubated, set the Plate Reader.
	<ol>
		<li>Set the Temperature to 37 &#176;C.</li>
		<li>In Settings, select Flex.</li>
		<li>Set the Read Mode to Fluorescence and Bottom read.</li>
		<li>In Wavelengths, set the number of Wavelengths to 2. Set the Excitation to 340 nm and 380 nm. Set the Emission to 510 nm.</li>
		<li>Leave the Sensitivity to Default.</li>
		<li>In Timing, set the Interval to 3.9 s. Set Run Time to 94 s to get 25 reads.</li>
		<li>Next, select the Assay Plate Type.</li>
		<li>Next, select the Wells to Read.</li>
		<li>In Compound Transfer, set Transfers to 1 and Initial Volume to 100 &#181;L. Set the Pipette Height to 100 &#181;L, Volume to 50 &#181;L, and Time Point to 36 s, to add the compound at the 10th reading.</li>
		<li>Next, select the Compound Source plate type.</li>
		<li>Leave Triturate to Not Used.</li>
		<li>Select the tips in the Pipette Tips Layout.</li>
		<li>For Compound and Tip Columns, ensure that the compounds to be transferred are in column 1 of the compound plate. Set the Tip Column to 1 and Compound Column to 1.</li>
		<li>Leave the Autocalibrate as ON.</li>
		<li>Click OK.</li>
	</ol>
	</li>
	<li>When the temperature has reached to 37 &#176;C, load the plates into the Plate Reader.
	<ol>
		<li>Press the Reading Chamber to put the Assay Plate into the fluorescence Plate Reader.</li>
		<li>Press the Source to put the Compound Plate. Prepare the Compound Plate by adding 200 &#181;L of respective peptides, ionomycin and ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)-Triton X-100 solutions.</li>
		<li>Press the Tip Rack to put the Tip Box. Use a black tip to avoid tip autofluorescence.</li>
		<li>Once the plates are loaded, review the settings of the software and press Read.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Determine the calcium concentration from the fluorescence ratio by the Grynkiewicz equation - 
	<img alt="Equation 1" src="/files/ftp_upload/62448/62448eq01.jpg" /> 
	where, Kd is the dissociation constant of Fura-2 AM, R is the emission ratio after excitation at 340 nm and 380 nm (F340/F380) for respective peptides, Rmax is the maximum fluorescence ratio observed by the addition of 50 &#181;L of 30 &#181;M ionomycin, Rmin is the minimum fluorescence observed by the addition of 50 &#181;L of 100 mM EGTA/2.5%Triton X-100, and F380min and F380max are the absolute fluorescence intensity of Fura-2 AM in calcium free and bound state, respectively. 
	NOTE: The machine dispenses the liquid with some force. Do not set the peptide dispensing height too close to the bottom of the plate; it may detach the cells. Use black pipette tips to avoid autofluorescence. Read the maximum fluorescence and the minimum fluorescence for each plate for each experiment.</li>
</ol>

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M., Kuehn, H. S., Kirshenbaum, A., Gilfillan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+isolation,+and+maintenance+of+human+mast+cells+and+mast+cell+lines+derived+from+peripheral+blood+or+cord+blood.">Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood.</a> Current protocols in immunology. , (2010).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+A+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: A vehicle for the expression of recombinant proteins.</a> Journal of Pharmacological and Toxicological Methods. 51 (3), 187-200 (2005).</li><li>Subedi, G. P., Johnson, R. W., Moniz, H. A., Moremen, K. 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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=Store-operated+calcium+entry+via+STIM1+contributes+to+MRGPRX2+induced+mast+cell+functions.">Store-operated calcium entry via STIM1 contributes to MRGPRX2 induced mast cell functions.</a> Frontiers in Immunology. 10, 3143 (2020).</li><li>Baba, Y., 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=Essential+function+for+the+calcium+sensor+STIM1+in+mast+cell+activation+and+anaphylactic+responses.">Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses.</a> Nature Immunology. 9 (1), 81-88 (2008).</li><li>Hoth, M., Penner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+intracellular+calcium+stores+activates+a+calcium+current+in+mast+cells.">Depletion of intracellular calcium stores activates a calcium current in mast cells.</a> Nature. 355 (6358), 353-356 (1992).</li><li>Assinger, A., Volf, I., Schmid, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+rapid+method+to+quantify+intraplatelet+calcium+dynamics+by+ratiometric+flow+cytometry.">A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.</a> PLoS One. 10 (4), 0122527 (2015).</li><li>Roe, M. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Generation+of+Ca2+++Indicators+with+Greatly+Improved+Fluorescence+Properties.">A New Generation of Ca2 + Indicators with Greatly Improved Fluorescence Properties.</a> Journal of Biological Chemistry. 260 (6), 3440-3450 (1985).</li><li>Lu, L., Parmar, M. B., Kulka, M., Kwan, P., Unsworth, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide+nanoscaffold+that+activates+human+mast+cells.">Self-assembling peptide nanoscaffold that activates human mast cells.</a> ACS Applied Materials and Interfaces. 10 (7), 6107-6117 (2018).</li><li>Lansu, 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=In+silico+design+of+novel+probes+for+the+atypical+opioid+receptor+MRGPRX2.">In silico design of novel probes for the atypical opioid receptor MRGPRX2.</a> Nature Chemical Biology. 13 (5), 529-536 (2017).</li><li>Navin&#233;s-Ferrer, 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=MRGPRX2-mediated+mast+cell+response+to+drugs+used+in+perioperative+procedures+and+anaesthesia.">MRGPRX2-mediated mast cell response to drugs used in perioperative procedures and anaesthesia.</a> Scientific Reports. 8 (1), 11628 (2018).</li><li>Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+store-operated+calcium+(Ca+2+)+entry+(SOCE)+in+HEK293+cells+using+Fura-2.">Calcium imaging of store-operated calcium (Ca 2+) entry (SOCE) in HEK293 cells using Fura-2.</a> Calcium Signalling. , 163-172 (2019).</li><li>Tinning, P. 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Authors declare no competing interests.

Calcium signaling is central to mast cell degranulation and has been widely used in the study of receptor-ligand interactions, ligand identification, and drug discovery14. MRGPRX2 is a recently discovered mast cell receptor that has been found to play a key role in many inflammatory diseases like itch, asthma, and atopic dermatitis, among others2. Furthermore, several approved drugs have been shown to elicit an inflammatory response through the MRGPRX2 receptor15. It is, therefore, imperative to study the ligand-receptor interaction, identify new ligands, and understand the activation mechanism. This study shows the use of common peptide techniques in designing a peptide library (Table 1) and in studying the MRGPRX2 based mast cell activation. The underlying calcium mobilization was employed as the indicator of mast cell activation. 
Fura-2 is a calcium sensitive dye used to measure intracellular calcium concentration. The acetoxymethyl ester variant (Fura-2 AM) increases the permeability of the cell membrane yielding an easy method for quantifying the cytoplasmic calcium releases. The dye has frequently been used with various characterization techniques like flow cytometer, microscopy, and fluorimeters9,16,17. Though widely used, there are challenges associated with the dye, which need to be addressed before these can be efficiently employed. Intracellular calcium binding relies on the de-esterification of the dye, which is largely dependent on the dye loading conditions, cell system, and cell culture types. Partial de-esterification results in inadequate fluorescence signals. Furthermore, incomplete de-esterification also results in the localization of dye into the cell organelles resulting in inaccurate data. Leakage of de-esterified dye from the cell is another issue faced with Fura-210,11. 
Apart from the dye loading, the use of the detection technique also plays an important role. The inability of flow cytometers to operate with UV lasers makes them unfavorable for the dye9. Similarly, fluorescent imaging techniques require advanced training in microscope operation. The transient nature of calcium release upon ligand activation requires a rapid response in the change in wavelengths and thus a faster shutter speed of the microscope. In addition, the use of specialized cell imaging chambers, use of coverslips and constant image focusing makes it a cumbersome technique for large-scale screening16.
A significant amount of time was invested in the method optimization. Cuvette based fluorometer, wherein cells after detaching from the culture flask, were incubated with the dye-loaded medium in the dark and then were washed and resuspended in the HTB buffer for the study. Cells suspended in HTB buffer were taken in a cuvette and were read using a photomultiplier based fluorometer. The detection systems could only hold few (2-4) cuvettes at a time and thus was not suitable for the large-scale screening of peptides. Further, HEK cells being adherent cells, the cell suspension reading showed great variations within individual repeats within an experiment. Consequently, fluorescent imaging technique was used, which again proved to be tedious and slow. The requirement for an advanced microscope with fast wavelength changing capability, microscope-specific cell chambers, and excellent imaging skills impeded the study. Additionally, in situ peptide simulation was time extensive and resulted in the loss of important data. Inefficient cell processing technique resulted in cell flotations during readings which made it difficult to focus and gave inconsistent results. 
A fluorescence plate reader system with an automated pipetting system that can dispense compounds during experiments was used for the study. The fact that many samples are read in quick succession in a 96 well plate is an added benefit of this technique. The results showed that the readings were more consistent when cells were attached as compared to suspension. Cell counts of 40,000 cells/well in a TC-treated 96 well plate grown for a day gave the best results. A dye incubation time of 30-40 min at 37 &#176;C incubator was optimum. However, when experiments were done with experimental repeats, variations in the corresponding wells of different columns were observed. To overcome this, a positive (PAMP-12) and a negative control (Blank) for each column of the plate was used. This gave more consistent and reproducible data. The method described here is an easy and versatile method, which can be efficiently employed for large-scale calcium-based screening. However, there are several factors which determines the quality of data and thus the method needs to be optimized for a given cell-instrument system.

Mast cells are an integral part of the immune system and play a crucial role in both innate and adaptive immune responses. Mast cells are primarily activated either by the binding of an antigen to the immunoglobulin E (IgE) - Fc&#949;RI receptor complex, or by the recently discovered mas related G-protein receptor-X2 (MRGPRX2)1. MRGPRX2 activation has been linked to several immune and inflammatory diseases, and hence, it is important to understand the binding mechanism of the receptor to its ligands2. To do so, a library of small peptide molecules was developed and screened against MRGPRX2 receptors that were overexpressed in HEK cells. In the study, the peptide library was constructed using the simple and versatile techniques of alanine scanning and amino acid truncation. Alanine scanning involves replacing specific amino acids with an alanine residue. Alanine being small and neutral, strips the peptide of the specific properties conferred by the replaced residue and consecutively highlights the significance of the respective physiochemical properties of the amino acid in receptor interactions. On the contrary, in amino acid truncation, peptide sequences are designed such that it lacks one or more amino acid residues from the N-terminal, C terminal, or both. This set of peptides was used to identify the amino acid sequences crucial to MRGPRX2 binding.
Experience with human mast cells lines (LAD-2) has shown that these cells are difficult to culture and maintain in vitro: a doubling time of two weeks, expensive medium supplements, and direct attention required during passaging3. These attributes make the cells unsuitable for large scale screening of potential ligands. Herein, stably transfected HEK cells expressing MRGPRX2 receptor (HEK-X2) were used to screen the peptide library1. HEK-293 cells are widely used and studied for the heterologous expression of surface receptors due to their high transfection efficiency, faster doubling rate, and the need for non-expensive medium supplements to be cultured in laboratory4. The protocol to transfect HEK-293 cell line has been demonstrated and is well established5. HEK-293 cells stably expressing MRGPRX2 receptor (passage 13-19) were activated with the peptides generated through N-truncation, C-truncation, N+C-truncation, and alanine scanning1. Wild type HEK cells (HEK-WT) (passage 16-21) were used as control. Intracellular calcium release upon activation was monitored to study the MRGPRX2 based activation.
Cell activation by MRGPRX2 is followed by a cytosolic calcium mobilization. This regulated intracellular calcium release in mast cells is regulated by the store operated calcium entry (SOCE), coordinated by the stromal interaction molecule 1 (STIM1); and is central to the immune response cascade6,7. Various methods have been used to detect intracellular calcium concentration, including patch-clamps and fluorescent dyes8. Of all the techniques available, fluorometric calcium dyes in conjugation with various detection techniques are being widely used9. Two types of fluorometric dyes that have gained interests are namely, single wavelength dyes like Fluo-4, and dual wavelength ratiometric dyes like Indo -1 and Fura-2. The advantage that dual wavelength ratiometric dyes bring over single wavelength dyes is that the ratiometric dyes correct for experimental errors like dye loading, photo bleaching, and focusing10,11.
Fura-2 acetoxymethyl ester (Fura-2 AM) is a cell permeating, green-fluorescent dye whose excitation shifts to a lower wavelength upon calcium-binding. Experimentally, Fura-2 is excited at 340 and 380 nm, while the emission is recorded at 510 nm. Upon calcium binding, the fluorescent intensity at 340 nm increases while that of 380 nm decreases, as shown in Figure 1. Data is represented as a ratio of fluorescence intensity after excitation at 340 nm (F340) to that of intensity after excitation at 380 nm (F380) i.e., F340/F380. The F340/F380 ratio is proportional to intracellular calcium, the value of which can be calculated by the Grynkiewicz equation12. Since the fluorescence signal is obtained from the excitation of the dye at two wavelengths (340 nm and 380 nm), the ratio of the fluorescence signals corrects for experimental factors like dye loading, dye leakage, photobleaching, and cell densities.

<table><tbody><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>5470</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma Aldrich</td><td>793939</td><td></td></tr><tr><td>Corning 96 Well</td><td>Sigma Aldrich</td><td>CLS3603</td><td></td></tr><tr><td>Black Polystyrene Microplate</td><td>Sigma Aldrich</td><td>CLS3603</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fischer</td><td>11995065</td><td>High Glucose</td></tr><tr><td>DMSO</td><td>Thermo Fischer</td><td>D12345</td><td>Sterile, biological grade</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E3889</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fischer</td><td>12483-020</td><td></td></tr><tr><td>Flexstation 3</td><td>Molecular devices</td><td>FV06060</td><td></td></tr><tr><td>Fura-2 AM</td><td>Thermo Fischer</td><td>F1221</td><td></td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>D8270</td><td></td></tr><tr><td>HEPES buffer</td><td>Thermo Fischer</td><td>15630-080</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma Aldrich</td><td>I9657</td><td></td></tr><tr><td>L Glutamine</td><td>Thermo Fischer</td><td>25030-081</td><td></td></tr><tr><td>Pen Strep</td><td>Thermo Fischer</td><td>15140122</td><td></td></tr><tr><td>Peptides</td><td>RS Syntehsis</td><td>Custom</td><td>&amp;ge;95% pure;&lt;br/&gt; N terminal - acetyl group&lt;br/&gt; C terminal - amide group</td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>12636</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S9888</td><td></td></tr><tr><td>TritonX-100</td><td>DOW Chemical</td><td>166704</td><td></td></tr></tbody></table>

screening peptides that activate mrgprx2 using engineered hek cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Table 1 contains the peptide sequences generated through terminal amino acid truncation and alanine scanning. As shown in Table 1, peptide sequence RKKWNKWALSR lacks N-terminal phenylalanine (F) with respect to its parent PAMP-12 and hence is a representative peptide in N-truncated library. Similarly, in FRKKWNKWALS, PAMP-12 C-terminal serine has been removed, representing a C-truncated peptide library derived from PAMP-12. In N+C-truncated peptide library, amino acid from both N and C-terminal are removed. Truncation of 4 amino acid from N-terminal and 1 residue from C-terminal of PAMP-12 results in WNKWALS. Peptide library derived from alanine scanning has one amino acid replaced with alanine, as with ARKKWNKWALSR, where N-terminal phenylalanine is replaced with alanine. Fura-2 AM dye was used to study the activation potential of peptides against MRGPRX2 transfected HEK cells1. The data was recorded on a fluorescence plate reader. If the peptide ligand activated the cell, the fluorescence pumped at 340 nm (F340) increases, while the same decreases for 380 nm wavelength (F380) (Figure 1a). For a blank (or for that matter non activating peptide or HEK-WT control) however, the relative increase and decrease would be respectively low, as shown in the Figure 2a. Calcium concentration is, however, represented by F340/F380 ratio as shown in Figure 1b,2b. The ratio F340/F380 can be further substituted in the Grynkiewicz equation to get the calcium concentration using in situ calibrations (Figure 3). The peptides listed in Table 1 were characterized by mass spectroscopy (Figure 4a) and HPLC (Figure 4b) and found to be of high purity.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peptide Technique</td>
			<td>Representative Peptide</td>
		</tr>
		<tr>
			<td>Parent peptide</td>
			<td>Ac-FRKKWNKWALSR-Amide</td>
		</tr>
		<tr>
			<td>N-Truncation</td>
			<td>Ac-RKKWNKWALSR-Amide</td>
		</tr>
		<tr>
			<td>C-Truncation</td>
			<td>Ac-FRKKWNKWALS-Amide</td>
		</tr>
		<tr>
			<td>N+C-Truncation</td>
			<td>Ac-WNKWALS-Amide</td>
		</tr>
		<tr>
			<td>Alanine Scanning</td>
			<td>Ac-ARKKWNKWALSR-Amide</td>
		</tr>
	</tbody>
</table>
Table 1: Representative peptide sequences generated after N, C and N+C - truncation, and alanine scanning. The N-terminal of the peptides were acetyl modified while the C-Terminal contained an amide group.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62448/62448fig01.jpg" /> 
Figure 1: Representative data for an activating peptide. (a) The fluorescence signals for an activating peptide. Represented data corresponds to PAMP-12 (FRKKWNKWALSR). Peptide was added after generating a baseline for 10 reading cycles (36 s), as shown by the arrow. (b) The ratio of the fluorescence emission after excitation at 340 nm (F340) to that of fluorescence emission after excitation at 380 nm (F380) (F340/F380). <a href="https://www.jove.com/files/ftp_upload/62448/62448fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62448/62448fig02.jpg" /> 
Figure 2: Representative data for the blank. (a) The fluorescence signals for the Blank. HTB was added after generating a baseline for 10 reading cycles (36 s), as shown by the arrow. (b) The ratio of the fluorescence emission after excitation at 340 nm (F340) to that of fluorescence emission after excitation at 380 nm (F380) (F340/F380). <a href="https://www.jove.com/files/ftp_upload/62448/62448fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62448/62448fig03.jpg" /> 
Figure 3: Representative data for the standards for dye calibration. (a) Ionomycin was added at 10th readings, as shown by the arrow, to get maximum fluorescence in Ca+2 bound state. EGTA-Triton X-100 was added after 20 readings, as shown by the arrow, to get a minimum signal. (b) The ratio of the fluorescence emission after excitation at 340 nm (F340) to that of fluorescence emission after excitation at 380 nm (F380) (F340/F380). These values are further put in the Grynkiewicz equation to get the intracellular calcium concentration. <a href="https://www.jove.com/files/ftp_upload/62448/62448fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62448/62448fig04.jpg" /> 
Figure 4: Characterization of a representative peptide to confirm the sequence and purity. (a) The theoretical mass of the representative peptide sequence WNKWAL was 857.90 Da, which was shown by the m/z ratio in mass spectroscopy. (b) Peptide&#39;s purity of 99% as confirmed by HPLC. This peptide belongs to N+C-truncated peptide library. <a href="https://www.jove.com/files/ftp_upload/62448/62448fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

screening-peptides-that-activate-mrgprx2-using-engineered-hek-cells

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

2.4K Views.  University of Alberta. Vandaag zullen we een methode demonstreren om een peptidebibliotheek te screenen, tegen een onlangs ontdekte masterreceptor, mas-gerelateerde G-eiwitreceptor X2, ook bekend als MRGPRX2-receptor. Mestcellen zijn een integraal onderdeel van het immuunsysteem en spelen een cruciale rol in zowel aangeboren als adaptieve immuunresponsen. Mestcellen worden geactiveerd door de antigeengebonden Fc epsilon R1-receptor of door de recent ontdekte X2-receptor.Activering van oppervlaktegebonden X2-...

Video: Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Genetics

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

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 ...

Biochemistry

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 ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Biology

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred &#181;m, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

Neuroscience

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

Bioengineering

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

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