We thank Dr. Ben Nacev for advice on design of the photoaffinity labeling protocol, Dr. Wei Shi for synthesizing the itraconazole photoaffinity probe, Dr. Yongjun Dang for advice on affinity pull-down experiments, and other members of the J.O.L. laboratory for helpful comments and support. This work was supported in part by a PhRMA Foundation Fellowship in Pharmacology/Toxicology (to S.A.H.); National Cancer Institute Grant R01CA184103; the Flight Attendant Medical Research Institute; Prostate Cancer Foundation (J.O.L.); and the Johns Hopkins Institute for Clinical and Translational Research, which is funded in part by Grant UL1 TR 001079 from the National Center for Advancing Translational Sciences (NCATS).

NOTE: This protocol was adapted from MacKinnon and Taunton10 for use in live cells.
1. Preparation of Cultured Cells
<ol>
	<li>Prepare sterile 6-cm cell culture dishes for the number of samples desired (see below). 
	NOTE: One dish of cells is used per treatment condition, but if more protein is required 2 or 3 dishes can be prepared per condition and combined after UV irradiation.
	<ol>
		<li>Prepare at least 3 dishes of cells; Negative control with DMSO only (D), Probe trea...

<ol>
	<li>Schenone, M., Dan&#269;&#236;k, V., Wagner, B. K., Clemons, 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=Target+identification+and+mechanism+of+action+in+chemical+biology+and+drug+discovery.">Target identification and mechanism of action in chemical biology and drug discovery.</a> Nat Chem Biol. 9 (4), 232-240 (2013).</li><li>Cook, D., Brown, D., 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=Lessons+learned+from+the+fate+of+AstraZeneca's+drug+pipeline:+a+five-dimensional+framework.">Lessons learned from the fate of AstraZeneca's drug pipeline: a five-dimensional framework.</a> Nat Rev Drug Discov. 13 (6), 419-431 (2014).</li><li>Titov, D. V., Liu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+protein+targets+of+bioactive+small+molecules.">Identification and validation of protein targets of bioactive small molecules.</a> Bioorg Med Chem. 20 (6), 1902-1909 (2012).</li><li>Jung, H. J., Kwon, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+deconvolution+of+bioactive+small+molecules:+the+heart+of+chemical+biology+and+drug+discovery.">Target deconvolution of bioactive small molecules: the heart of chemical biology and drug discovery.</a> Arch Pharm Res. 38 (9), 1627-1641 (2015).</li><li>Sumranjit, J., Chung, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Advances+in+Target+Characterization+and+Identification+by+Photoaffinity+Probes.">Recent Advances in Target Characterization and Identification by Photoaffinity Probes.</a> Molecules. 18 (9), 10425-10451 (2013).</li><li>Robles, O., Romo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemo-+and+site-selective+derivatizations+of+natural+products+enabling+biological+studies.">Chemo- and site-selective derivatizations of natural products enabling biological studies.</a> Nat Prod Rep. 31 (3), 318-334 (2014).</li><li>Zhou, Q., Gui, 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=Bioconjugation+by+Native+Chemical+Tagging+of+C-H+Bonds.">Bioconjugation by Native Chemical Tagging of C-H Bonds.</a> J Am Chem Soc. 135 (35), (2013).</li><li>Li, Z., Hao, P., 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=Design+and+Synthesis+of+Minimalist+Terminal+Alkyne-Containing+Diazirine+Photo-Crosslinkers+and+Their+Incorporation+into+Kinase+Inhibitors+for+Cell-+and+Tissue-Based+Proteome+Profiling.">Design and Synthesis of Minimalist Terminal Alkyne-Containing Diazirine Photo-Crosslinkers and Their Incorporation into Kinase Inhibitors for Cell- and Tissue-Based Proteome Profiling.</a> Angew Chem Int Edit. 52 (33), 8551-8556 (2013).</li><li>Chamni, S., He, Q. 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L., Taunton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+Identification+by+Diazirine+Photo-Cross-linking+and+Click+Chemistry.">Target Identification by Diazirine Photo-Cross-linking and Click Chemistry.</a> Curr Protoc Chem Biol. 1, 55-73 (2009).</li><li>Smith, E., Collins, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoaffinity+labeling+in+target-+and+binding-site+identification.">Photoaffinity labeling in target- and binding-site identification.</a> Future med chem. 7 (2), 159-183 (2015).</li><li>Huisgen, R., Szeimies, G., M&#246;bius, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1.3-Dipolare+Cycloadditionen,+XXXII.+Kinetik+der+Additionen+organischer+Azide+an+CC-Mehrfachbindungen.">1.3-Dipolare Cycloadditionen, XXXII. Kinetik der Additionen organischer Azide an CC-Mehrfachbindungen.</a> Chem Ber. 100 (8), 2494-2507 (1967).</li><li>Kolb, H. C., Finn, M. G., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry:+Diverse+Chemical+Function+from+a+Few+Good+Reactions.">Click Chemistry: Diverse Chemical Function from a Few Good Reactions.</a> Angew Chem Int Ed Engl. 40 (11), 2004-2021 (2001).</li><li>Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+huisgen+cycloaddition+process:+copper(I)-catalyzed+regioselective+&amp;#34;ligation&amp;#34;+of+azides+and+terminal+alkynes.">A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective &amp;#34;ligation&amp;#34; of azides and terminal alkynes.</a> Angew Chem Int Ed Engl. 41 (14), 2596-2599 (2002).</li><li>Torn&#248;e, C. W., Christensen, C., Meldal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidotriazoles+on+Solid+Phase:+[1,2,3]-Triazoles+by+Regiospecific+Copper(I)-Catalyzed+1,3-Dipolar+Cycloadditions+of+Terminal+Alkynes+to+Azides.">Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides.</a> J Org Chem. 67 (9), 3057-3064 (2002).</li><li>Head, S. A., Shi, W., 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=Antifungal+drug+itraconazole+targets+VDAC1+to+modulate+the+AMPK/mTOR+signaling+axis+in+endothelial+cells.">Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells.</a> P Natl Acad Sci USA. 112 (52), E7276-E7285 (2015).</li><li>Shi, W., Nacev, B. A., Aftab, B. T., Head, S., Rudin, C. M., Liu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itraconazole+Side+Chain+Analogues:+Structure-Activity+Relationship+Studies+for+Inhibition+of+Endothelial+Cell+Proliferation,+Vascular+Endothelial+Growth+Factor+Receptor+2+(VEGFR2)+Glycosylation,+and+Hedgehog+Signaling.">Itraconazole Side Chain Analogues: Structure-Activity Relationship Studies for Inhibition of Endothelial Cell Proliferation, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, and Hedgehog Signaling.</a> J Med Chem. 54 (20), 7363-7374 (2011).</li><li>Shi, W., Nacev, B. A., Bhat, S., Liu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Absolute+Stereochemistry+on+the+Antiangiogenic+and+Antifungal+Activities+of+Itraconazole.">Impact of Absolute Stereochemistry on the Antiangiogenic and Antifungal Activities of Itraconazole.</a> ACS Med Chem Lett. 1 (4), 155-159 (2010).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> J Biol Chem. 193 (1), 265-275 (1951).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Bioph Res Co. 28 (5), 815-820 (1967).</li><li>Switzer, R. C., Merril, C. R., Shifrin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+silver+stain+for+detecting+proteins+and+peptides+in+polyacrylamide+gels.">A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.</a> Anal Biochem. 98 (1), 231-237 (1979).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> P Natl Acad Sci USA. 76 (9), 4350-4354 (1979).</li></ol>

The authors have nothing to disclose.

Different approaches to identifying the targets of small molecules can be broadly grouped into two categories: top-down, where the cellular phenotype of the drug is used to narrow down its potential targets based on their known functions, or bottom-up, where the target is identified directly by chemical or genetic means3. Top-down or phenotypic studies can identify certain cellular processes affected by the drug (e.g., transcription/translation/DNA synthesis, cell cycle block, signaling pathway activa...

Bioactive small molecules fundamentally work by interacting with and altering the function of one or more &#34;target&#34; molecules, most commonly proteins, in the cell. In drug discovery, when an active compound is discovered through phenotypic screening, identification of the molecular target(s) of that compound is crucial, not just for understanding the mechanism of action and potential side-effects of the compound, but also for potentially discovering new biology underlying the disease model and paving the way for development of new mechanistic classes of therapeutics1. Although target identification is not required for a drug to be used therapeutically, in recent years there has been an increasing recognition that novel drug candidates are more likely to succeed in clinical trials, and therefore yield better returns on investment, if a validated target is known2. Thus, there has been a growing interest in methods for identifying small molecule target proteins.
A classical target identification experiment typically relies on affinity purification, where the small molecule of interest is immobilized onto a resin and incubated with whole cell lysates, after which unbound proteins are washed away and the remaining proteins are eluted and identified3. While this technique has been used to identify the targets of many small molecules4, it is unsuitable as a universal target ID method for several reasons. First, the target protein must retain its native conformation upon cell lysis in order to retain its ability to bind to the small molecule. This can be particularly problematic for membrane proteins, which often undergo conformational changes after being removed from their native environment, or simply aggregate and precipitate out of solution. Second, the small molecule must be chemically modified in such a way that it can be immobilized onto the resin while maintaining its ability to bind the target protein. Deep binding pockets may therefore become inaccessible to a small molecule once it is fixed to the resin. Third, the binding affinity must be sufficiently high that the interaction is maintained during the washing steps, making identification of lower affinity interactions challenging. Fourth, environmental conditions such as pH, ion concentration, or the presence of other endogenous molecules can vary spatially within the cell and are sometimes prerequisites for drug-target interactions. Thus, finding the correct conditions to allow and maintain binding outside of the cell can require a significant amount of trial and error.
Photoaffinity labeling circumvents these issues by allowing the covalent binding of a small molecule and its target within the native context of a cell. Rather than immobilizing the small molecule to a large bulky resin, the molecule is instead chemically modified to install two small functional groups: a photoactivatable moiety which allows covalent crosslinking to the target protein when irradiated with a particular wavelength of light, and a reporter group that allows the target protein to be detected and subsequently isolated. Live cells are treated with the photoaffinity probe, the probe binds and covalently crosslinks to the target protein, and the probe-protein complex is then isolated intact. The specificity of probe binding to the target is demonstrated by performing a competition experiment in parallel, where an excess of the parent compound is used to compete away binding of the probe to the target protein.
The design and synthesis of photoaffinity probes varies greatly from one small molecule to another, and will not be covered in this protocol; however, several excellent discussions on the subject have been published5-9. The main consideration is that the probe retains the bioactivity of the parent compound, therefore presumably binding to the same target(s). Structure-activity relationship (SAR) studies must be performed to determine which parts of the molecule can be modified without loss of bioactivity. A variety of different chemical groups have been used as photoactivatable crosslinkers, including diazirine, benzophenone, and aryl azide, which each have advantages and disadvantages10. Likewise, there are multiple reporter tags that have been used to isolate probe-binding proteins. Reporter groups may be functional on their own, such as the commonly used biotin or fluorescent tags, or may be precursors that require further functionalization subsequent to the photocrosslinking step, which have the advantage of being smaller and thus less likely to compromise bioactivity11.
In this protocol, we have used a photoaffinity probe containing a diazirine photocrosslinking group, and a terminal alkyne for the attachment of a reporter group through a Cu(I)-catalyzed Azide-Alkyne Sharpless-H&#252;isgen cycloaddition (or click) reaction12-15. The SAR studies, probe design and synthesis, and results of these studies have been published elsewhere16-18.

<table border="0" cellpadding="0" cellspacing="0" width="1169">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td style="width:188px;">Life Technologies</td>
			<td style="width:117px;">20490</td>
			<td style="width:481px;">Make fresh day of use. Prepare 100 mM stock in water with 4 eq NaOH.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA)&#160;</td>
			<td>AnaSpec</td>
			<td>63360-50&#160;</td>
			<td>Prepare 1.7 mM stock in a 4:1 ratio of t-butanol to DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Copper Sulfate (CuSO4-5H2O)</td>
			<td>LabChem, Inc.</td>
			<td>LC13440-1&#160;</td>
			<td>Prepare 50 mM stock in water, store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin-azide</td>
			<td>Click Chemistry Tools</td>
			<td>AZ104-100</td>
			<td>Prepare 10 mM stock in DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647-azide&#160;</td>
			<td>Life Technologies</td>
			<td>A10277</td>
			<td>Prepare 1 mM stock in DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">365 nm UV lamp</td>
			<td>Spectroline</td>
			<td>FC100</td>
			<td>UV-blocking glasses should be worn while operating.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor tablets, EDTA-free</td>
			<td>Roche Life Science</td>
			<td>11873580001</td>
			<td>Prepare 50x solution in water and store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>Branson</td>
			<td>Sonifier 250</td>
			<td>Set to output 1, duty 30%.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent gel scanner</td>
			<td>GE Healthcare Life Sciences</td>
			<td>FLA 9500</td>
			<td>Use red laser to detect Alexa-fluor 647.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Detergent-compatible Dc protein assay kit</td>
			<td>Bio-Rad</td>
			<td>5000112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Capacity Streptavidin Agarose beads&#160;</td>
			<td>Life Technologies</td>
			<td>20359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagles Medium, low glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>11885092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, qualified</td>
			<td>ThermoFisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptamycin solution</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS Sample Buffer (2X)</td>
			<td>ThermoFisher Scientific</td>
			<td>LC2676</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

The results shown here were obtained with a photo-affinity probe of the antifungal drug itraconazole, the use of which has been previously published16. These results demonstrate the use of the live-cell photoaffinity labeling technique to successfully identify a major itraconazole-binding protein as the 35 kDa membrane protein Voltage-Dependent Anion Channel 1 (VDAC1).
The above protocol was performed in HEK293T cells...

Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

The overall goal of this procedure, is to identify the binding proteins of small molecules by using a photoaffinity probe, that can bind to its target in live cells, allowing the subsequent isolation and identification of the target. This method can be used to elucidate the molecular mechanism of action of drugs or other small molecules. The main advantage of this technique, is that binding and covalent labeling of the target proteins occur within the native cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.Begin this procedure, by preparing sterile six centimeter cell culture dishes, for the number of samples desired. Typically, one dish of cells is used per treatment condition. Three dishes of cells will be prepared for this demonstration, and negative control with DMSO only, labeled D, probe treatment only, labeled P, and probe plus competitor, labeled C.To each culture dish, add 3.5 million HEK 293 T cells in four milliliters of culture medium.Incubate the cells overnight. Prior to treating the cells, pre-aliquot the needed drugs into 1.5 milliliter microcentrifuge tubes. For the competitor pre-treatment, add four microliters of DMSO to each of two tubes, and four microliters of 10 millimolar competitor to one tube.For the probe treatment, add 16 microliters of DMSO to one tube, and 16 microliters of 15 micromolar photoaffinity probe to each of two tubes. Bring the cell culture dishes into the cell culture hood for the addition of the pre-aliquoted drugs. Aspirate one milliliter of culture medium from the competition treatment dish, transfer it to the microcentrifuge tube that contains the pre-aliquoted competitor, and resuspend the drug in the medium.Gently add the medium and drug mixture dropwise back to the dish. In the same manner, add DMSO to both the negative control dish and the probe dish. Return the dishes to the incubator for 30 minutes.After 30 minutes, bring the dishes back to the culture hood and dim the lights. Add the pre-aliquoted DMSO to the negative control dish, and probe to the probe and competition dishes. Return the dishes to the incubator for one hour.After one hour, place the dishes on ice. Wash the cells in each dish gently with five milliliters of ice cold PBS to remove excess probe. Re-cover the cells with four milliliters of ice cold PBS.Place a dish of cells, centered three centimeters under the UV lamp on top of an ice pack, to minimize heating from the lamp, and irradiate for three minutes. Remove the dish and place it on ice. In this manner, irradiate all the samples.After irradiation of all samples, aspirate the PBS from the cells, and add 200 microliters of ice cold PBS with protease inhibitors to each dish. Detach the cells from the dish using a rubber scraper, and transfer to pre-labeled microcentrifuge tubes on ice. Add SDS to each sample to a final concentration of 0.4%Lyse the cells by sonicating the suspension for 10 pulses, incubating on ice for one minute, and then sonicating for another 10 pulses.Boil the samples on a heat block set to 95 degrees Celsius for five minutes to complete cell lysis and denature all the proteins. After measuring the protein concentration in each sample, as described in the text protocol, normalize the protein concentration to 2.5 milligrams per milliliter, by adding PBS PH 8.5 plus 0.4%SDS as needed. To begin this procedure, transfer 40 microliters of each cell lysate, prepared in the previous segment to a new microcentrifuge tube.Add the following reagents in this order, 0.2 microliters of flour-azide, 0.58 microliters of TCEP, and 3.38 microliters of TBTA. Vortex to mix. Add 1.14 microliters of copper sulfate pentahydrate to start the reaction.Vortex briefly and incubate at room temperature for 30 minutes in the dark. Add 50 microliters of 2X SDS sample buffer to quench the reaction. Run the samples on an SDS-PAGE gel, and when the dye front has reached the end of the gel, continue running the gel for additional five minutes to ensure all of the excess unreacted flour-azide has completely exited the gel.After washing away all excess flour-azide, place the gel onto a glass plate and scan the gel using a fluorescent gel scanner, according to the manufacturer's instructions. For the attachment of a biotin tag, use the maximum amount of cell lysates after protein normalization, such that all samples are of the same volume. Preclear the lysates by adding each sample to a new tube, containing 50 microliters of pre-washed high capacity streptavidin agarose beads.Incubate for one hour at four degrees Celsius with rotation. Pellet the beads by centrifugation. Remove the supernatant to a new microcentrifuge tube on ice, and discard the beads.Per 500 microliters of lysate, add the following reagents, 1.38 microliters of biotin-azide, 5.5 microliters of TCEP, and 32.5 microliters of TBTA. Vortex to mix. Add 11 microliters of copper sulfate pentahydrate per 500 microliters of lysate and vortex briefly.Incubate at room temperature for 30 minutes. Add four sample volumes of acetone, chilled to 20 degrees Celsius, vortex the samples, and incubate overnight at 80 degrees Celsius, to completely precipitate the proteins and remove unreacted biotin-azide. On the following day, centrifuge the samples at 17, 000 x g for 15 minutes at four degrees Celsius to pellet the precipitated proteins.Aspirate the supernatant completely, add 150 microliters of PBS PH 7.4 and 1%SDS to each sample, and resoluablize the proteins by sonication. Add 600 microliters of PBS to each sample, to dilute the concentration of SDS to 0.2%then add the sample to a new tube containing 30 microliters of pre-washed high capacity streptavidin agarose beads. Incubate for one hour at four degrees Celsius with rotation.After one hour, pellet the beads by centrifugation at 1000 x g for three minutes. Aspirate and discard the supernatant, containing unbound proteins. Add one milliliter of wash buffer to the beads, and incubate for five minutes at room temperature with rotation.Centrifuge, discard the supernatant, and wash with wash buffer again. After the final wash, aspirate the wash buffer completely from the beads, and add 30 microliters of 2X SDS sample buffer. Incubate for five minutes in a 95 degrees Celsius heatblock, to release the proteins from the beads.Centrifuge the beads at 13000 x g at room temperature for one minute. Carefully pipette the sample buffer, containing proteins off of the beads, for SDS-PAGE. The visualization of proteins after labeling with the fluorescent tag, is illustrated by this fluorescent scanned gel.The major specific binding protein band, at approximately 35 kilodaltons, is only present in the probe lane, and is competed away by excess parent compound. The same 35 kilodalton band was visualized by silver staining after biotin pulldown, and subsequently identified by mass spectrometry as the membrane protein VDAC1. The identity of the protein was further validated, using a specific antibody for VDAC1.The signal is present in the probe lane, and decreased in the competition lane. Including the input fraction, ensures that the antibody works and the protein of interest can be detected in the lysate. The slight increase in molecular weight of the pulldown samples, is due to the covelantly attached probe.The consequences of not performing certain steps in the protocol correctly are illustrated. Incomplete removal of excess flour-azide, results in large black smears at the bottom of the fluorescent scanned gel, while insufficient preclearing of the lysates and or washing of the beads, results in a silver stained gel with very high background staining. From start to finish, the pulldown experiment takes two to three days to complete.After the target protein has been isolated and identified by mass spectrometry or western blotting, further target specific validation experiments should be performed to assess the relevance of the target to the drug-induced phenotype.

identification-small-molecule-binding-proteins-native-cellular

Research

JoVE Journal

Biochemistry

12.4K Görüntüleme.  Johns Hopkins University School of Medicine. The overall goal of this procedure, is to identify the binding proteins of small molecules by using a photoaffinity probe, that can bind to its target in live cells, allowing the subsequent isolation and identification of the target. This method can be used to elucidate the molecular mechanism of action of drugs or other small molecules. The main advantage of this technique, is that binding and covalent labeling of the target proteins occur within the native cellular environment, removing the...