Name: A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis (Video) | JoVE Journal
Uploaded: 2017-01-24T12:30:00.000+00:00
Duration: 5 min 43 s
Description: Learn a protein preparation method for identifying nuclear cofactor interactions using LC-MS/MS analysis in HEK 293 cells. This immunoprecipitation technique enables high-throughput discovery of transcriptional regulatory protein networks.

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5 minutes

This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), the Takeda Science Foundation (HT), and by a Japan Society for the Promotion of Science Grants-in-Aid for Young Scientists (B) to HT.

1. Transfection
<ol>
	<li>Seed HEK293 cells in 100-mm culture plates (2 x 106 cells/dish). Culture the cells in Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% fetal calf serum, 100 &#181;g/ml streptomycin, and 100 units/ml penicillin. Incubate the cells in a humidified incubator at 5% CO2 and 37 &#176;C.</li>
	<li>The next day, transfect the cells with 10 &#181;g of FLAG-tagged ARIP4 plasmid using 40 &#181;l of transfection reagent according to the manufacturer&#39;s directions11.</li>
</ol>
2. Protein Extraction
<ol>
	<li>Approximately 36 hr post-transfection, wash the cells with ice-cold PBS and harvest the cells using a scraper. Transfer the cells into a 1.5 mltube and centrifuge it at 8,000 x g for 2 min at 4 &#176;C. Aspirate the supernatant and resuspend the cell pellet in 5x the pellet volume of 0.4 M KCl buffer (High salt buffer: 20 mM Tris-HCl (pH 8.0), 0.4 M KCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 10 mM &#946;-mercaptethanol, 1x protease inhibitor cocktail). Rotate the mixture for 20 min at 4 &#176;C.</li>
	<li>Centrifuge the tube at 17,700 x g for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatant (whole-cell extract) to a new 2-mL tube and add 3x the initial volume of 0 M KCl buffer (Dilution buffer: 20 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 10 mM &#946;-Mercaptoethanol, 1x protease inhibitor cocktail).</li>
	<li>Perform another centrifugation (17,700 x g for 10 min at 4 &#176;C) and transfer the supernatant (whole-cell extract) to a new 2 ml tube.</li>
</ol>
3. Immunoprecipitation
<ol>
	<li>During the centrifugation (2.4), wash 50 &#181;l of anti-FLAG beads with PBS, 0.1 M glycine-hydrochloride, and then 1 M Tris-hydrochloride, followed by washing twice with 0.1M KCl buffer (Low salt buffer: 20 mM Tris-HCl (pH 8.0), 0.1 M KCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 10 mM &#946;-mercaptoethanol, 1x protease inhibitor cocktail). The centrifugation for this washing process is performed at 2,000 x g for 1 min at 4 &#176;C.</li>
	<li>Mix 50 &#181;l of FLAG beads with the whole-cell extracts and gently rotate the mixture for 4 hr at 2-4 &#176;C (1% of the whole-cell extract from step 2.4 should also be kept at -80 &#176;C for future use as an input).</li>
	<li>Transfer the sample to a micro spin column (gravity drop). Keep the flow-through in a 1.5 ml tube, freeze it using liquid nitrogen, and store it at -80 &#176;C (check the protein levels of both the input and flow-through using Western blot analysis).</li>
	<li>Add 1 ml of 0.1 M KCl buffer (Low salt buffer) to wash the FLAG beads (gravity drop).</li>
	<li>Repeat step 3.4 at least four more times (for a total of five washes).</li>
</ol>
4. Elution
<ol>
	<li>Place the column into a 1.5 ml tube and centrifuge it at 2,000 x g for 1 min; the beads will dry.</li>
	<li>Cap the bottom of the column.</li>
	<li>Add 50 &#181;l (equal to the volume of FLAG beads) of 0.1 M glycine-HCl (pH 2.5).</li>
	<li>Gently shake the mixture for 10 min at room temperature.</li>
	<li>Place the column into a new 1.5 ml tube and centrifuge it at 2,000 x g for 1 min.</li>
	<li>The eluted solution is neutralized with 10 &#181;l of 1 M Tris-HCl (pH 8.0); mix well by pipetting or by using a vortex machine.</li>
</ol>
5. Alkylation and Trypsinization
<ol>
	<li>Add 50 &#181;l of 100 mM NH4HCO3, 10 &#181;l of CH3CN, and 2 &#181;l of dithiothreitol to the mixture (112 &#181;l total volume).</li>
	<li>Incubate the sample for 30 min at 56 &#176;C.</li>
	<li>Place the sample at room temperature for 10 min.</li>
	<li>Add 10 &#181;l of 333 mM iodoacetamide to the sample and incubate it in the dark for 30 min at 37 &#176;C.</li>
	<li>Add 10 &#181;l of trypsin (33 ng/&#181;l) to the mixture and incubate it overnight at 37 &#176;C.</li>
</ol>
6. LC-MS/MS Analysis
<ol>
	<li>Following overnight trypsinization, transport the final product to an LC-MS/MS facility or to a third-party mass spectrometry lab for analysis12.</li>
</ol>

<ol><li>De Las Rivas, J., Fontanillo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-protein+interactions+essentials%3A+key+concepts+to+building+and+analyzing+interactome+networks%5BTitle%5D)+AND+%22PLoS+Comput.Biol%22%5BJournal%5D)">Protein-protein interactions essentials: key concepts to building and analyzing interactome networks.</a> PLoS Comput.Biol. 6 (6), e1000807(2010).</li>
<li>Westermarck, J., Ivaska, J., Corthals, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+protein+interactions+involved+in+cellular+signaling%5BTitle%5D)+AND+%22Mol.Cell.Proteomics%22%5BJournal%5D)">Identification of protein interactions involved in cellular signaling.</a> Mol.Cell.Proteomics. 12 (7), 1752-1763 (2013).</li>
<li>MacPherson, R. E., Ramos, S. V., Vandenboom, R., Roy, B. D., Peters, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Skeletal+muscle+PLIN+proteins%2C+ATGL+and+CGI-58%2C+interactions+at+rest+and+following+stimulated+contraction%5BTitle%5D)+AND+%22Am.J.Physiol.Regul.Integr.Comp.Physiol%22%5BJournal%5D)">Skeletal muscle PLIN proteins, ATGL and CGI-58, interactions at rest and following stimulated contraction.</a> Am.J.Physiol.Regul.Integr.Comp.Physiol. 304 (8), 644-650 (2013).</li>
<li>Qin, K., Dong, C., Wu, G., Lambert, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inactive-state+preassembly+of+G%28q%29-coupled+receptors+and+G%28q%29+heterotrimers%5BTitle%5D)+AND+%22Nat.Chem.Biol%22%5BJournal%5D)">Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers.</a> Nat.Chem.Biol. 7 (10), 740-747 (2011).</li>
<li>Ogawa, H., Ishiguro, K., Gaubatz, S., Livingston, D. M., Nakatani, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+complex+with+chromatin+modifiers+that+occupies+E2F-+and+Myc-responsive+genes+in+G0+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells.</a> Science. 296 (5570), 1132-1136 (2002).</li>
<li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordinated+histone+modifications+mediated+by+a+CtBP+co-repressor+complex%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Coordinated histone modifications mediated by a CtBP co-repressor complex.</a> Nature. 422 (6933), 735-738 (2003).</li>
<li>Huh, K. W., DeMasi, J., Ogawa, H., Nakatani, Y., Howley, P. M., Munger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+the+human+papillomavirus+type+16+E7+oncoprotein+with+the+600-kDa+retinoblastoma+protein-associated+factor%2C+p600%5BTitle%5D)+AND+%22Proc.Natl.Acad.Sci.U.S.A%22%5BJournal%5D)">Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600.</a> Proc.Natl.Acad.Sci.U.S.A. 102 (32), 11492-11497 (2005).</li>
<li>Huang, B. X., Kim, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effective+identification+of+Akt+interacting+proteins+by+two-step+chemical+crosslinking%2C+co-immunoprecipitation+and+mass+spectrometry%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Effective identification of Akt interacting proteins by two-step chemical crosslinking, co-immunoprecipitation and mass spectrometry.</a> PLoS One. 8 (4), 61430(2013).</li>
<li>ten Have, S., Boulon, S., Ahmad, Y., Lamond, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry-based+immuno-precipitation+proteomics+-+the+user%27s+guide%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Mass spectrometry-based immuno-precipitation proteomics - the user's guide.</a> Proteomics. 11 (6), 1153-1159 (2011).</li>
<li>Ogawa, H., Komatsu, T., Hiraoka, Y., Morohashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+Suppression+by+Transient+Recruitment+of+ARIP4+to+Sumoylated+nuclear+receptor+Ad4BP%2FSF-1%5BTitle%5D)+AND+%22Mol.Biol.Cell%22%5BJournal%5D)">Transcriptional Suppression by Transient Recruitment of ARIP4 to Sumoylated nuclear receptor Ad4BP/SF-1.</a> Mol.Biol.Cell. 20 (19), 4235-4245 (2009).</li>
<li>Tsuchiya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+autophagic+receptor+p62+regulates+the+abundance+of+transcriptional+coregulator+ARIP4+during+nutrient+starvation%5BTitle%5D)+AND+%22Sci.Rep%22%5BJournal%5D)">Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation.</a> Sci.Rep. 5, 14498(2015).</li>
<li>Watanabe, T., Matsuo, I., Maruyama, J., Kitamoto, K., Ito, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+an+intracellular+lectin%2C+calnexin%2C+from+Aspergillus+oryzae+using+N-glycan-conjugated+beads%5BTitle%5D)+AND+%22Biosci.Biotechnol.Biochem%22%5BJournal%5D)">Identification and characterization of an intracellular lectin, calnexin, from Aspergillus oryzae using N-glycan-conjugated beads.</a> Biosci.Biotechnol.Biochem. 71 (11), 2688-2696 (2007).</li>
<li>Sitz, J. H., Tigges, M., Baumgartel, K., Khaspekov, L. G., Lutz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dyrk1A+potentiates+steroid+hormone-induced+transcription+via+the+chromatin+remodeling+factor+Arip4%5BTitle%5D)+AND+%22Mol.Cell.Biol%22%5BJournal%5D)">Dyrk1A potentiates steroid hormone-induced transcription via the chromatin remodeling factor Arip4.</a> Mol.Cell.Biol. 24 (13), 5821-5834 (2004).</li>
<li>Mohammed, H., Taylor, C., Brown, G. D., Papachristou, E. K., Carroll, J. S., D'Santos, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+immunoprecipitation+mass+spectrometry+of+endogenous+proteins+%28RIME%29+for+analysis+of+chromatin+complexes%5BTitle%5D)+AND+%22Nat.Protoc%22%5BJournal%5D)">Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes.</a> Nat.Protoc. 11 (2), 316-326 (2016).</li>
<li>Fonslow, B. R., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capillary+electrophoresis+applied+to+proteomic+analysis%5BTitle%5D)+AND+%22J.Sep.Sci%22%5BJournal%5D)">Capillary electrophoresis applied to proteomic analysis.</a> J.Sep.Sci. 32 (8), 1175-1188 (2009).</li>
</ol>

The authors have nothing to disclose.

Efficient transfection is essential to obtain a successful result with this protocol. Accordingly, we recommend using Western blot analysis to determine the immunoprecipitated FLAG-tagged protein levels. This step enables users to verify that their protein of interest is properly overexpressed and, moreover, that the IP was performed successfully. FLAG-tagged protein levels should also be checked prior to the mass spectrometry analysis.
A mock sample should be used as a negative control to avo...

Protein-protein interactions play an important role in many biological functions. As such, these interactions have been implicated in signal transduction; protein transport across cell membranes; cell metabolism; and several nuclear processes, including DNA replication, DNA damage repair, recombination, and transcription1,2,3,4. Identifying the proteins involved in these interactions is therefore critical to advancing our understanding of these cellular processes.
Immunoprecipitation (IP) is a validated technique used to analyze protein-protein interactions. In order to facilitate the identification of co-immunoprecipitated proteins, mass spectrometry is often utilized5,6,7,8,9. By targeting a known member of a protein complex with an antibody, it is possible to isolate the protein complex and to subsequently identify its unknown components via mass spectrometry analysis. ARIP4 (androgen receptor-interacting protein 4), a transcriptional coregulator, interacts with nuclear receptor proteins in order to activate or repress its target promoters in a context-dependent manner9,10. To better understand the transcriptional regulatory mechanisms governing these nuclear factors, we describe a comprehensive method to purify and identify ARIP4 interacting proteins by using the LC-MS/MS system.

<table><tbody><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>Protease Inhibitor Cocktail (EDTA free) (100x)</td><td>nacalai tesque</td><td>03969-21</td><td></td></tr><tr><td>ANTI-FLAG M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>A2220</td><td></td></tr><tr><td>Micro Bio-Spin Columns</td><td>BIO-RAD</td><td>732-6204</td><td></td></tr></tbody></table>

a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We identified a strong ARIP4 signal, around 160 KDa, as well as those of a mock sample control and several other unknown proteins (Figure 1). LC-MS/MS analysis identified both the ARIP4 complex peptides and the potential peptide cofactors within the fraction of FLAG beads (Table 1). p62 (Sequestosome1), a known ARIP4 cofactor11 was identified in the analysis (Table 1), which confirms the efficacy of this system<sup...

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Overall goal of this procedure is to identify proteins interacting with a Nuclear Cofactor using LC-MS/MS Analysis. This method can help answer key questions related to transcriptional mechanisms in the gene regulation field. The main advantage of this technique is that we can identify the proteins interacting with a nuclear cofactor.Moreover, this method allows us to better understand the transcriptional regulatory mechanisms of the targeted nuclear factors. Harvest HEK 293 cells expressing flag-tagged ARIP4 36 hours after transvection by washing the cell sheet with ice cold PBS and collecting the cells with a cell scrapper. Transfer the cells into a 1.5 milliliter tube and then centrifuge at 8000g for two minutes at four degrees Celsius.Aspirate the supernatant and re-suspend the cell pellet in five times the pellet volume of High Salt Buffer. Rotate the mixture for 20 minutes at four degrees Celsius. After 20 minutes of rotation, centrifuge the tube at 17, 700g for 10 minutes at four degrees Celsius.Next, transfer the supernatants to a new two milliliter tube and add Dilution buffer to three times the volume of supernatant. Perform another cetrifugation and then transfer the supernatant to a new two milliliter tube. During the 10 minute centrifugation wash 50 microliters of anti-flag beads with PBS.Then centrifuge the tube at 2000g for one minute at four degrees Celsius. Remove the supernatant and repeat wash steps first with 1 molar glycine hydrochloride and then with one molar tris hydrochloride. Then wash twice with Low salt buffer.Next, mix 50 microliters of washed beads with whole-cell extracts. Gently rotate the mixture for four hours at two to four degrees Celsius. Then use a blunted tip to transfer the sample into a micro spin column and allow the solution to drip through into a 1.5 milliliter tube.Snap freeze the flow through in liquid nitrogen and store at minus 80 degrees Celsius. Next, wash the flag beads with Low salt buffer five times. To elute, place the column into a 1.5 milliliter tube and centrifuge at 2000g for one minute to dry the beads.Cap the bottom of the column and then add 50 microliters of 1 molar glycine hydrochloride. Gently shake the mixture for 10 minutes at room temperature. After 10 minutes, place the column into a new 1.5 milliliter tube and centrifuge at 2000g for one minute.Neutralize the eluted solution with 10 microliters of one molar tris hydrochloride and mix well by pipetting and vortexing. Add 50 microliters of 100 millimolar ammonium bicarbonate. 10 microliters of acetonitrile, two microliters of dithiothreitol and 50 microliters of neutralized sample.Incubate for 30 minutes at 56 degrees Celsius. After incubation, leave the sample at room temperature for 10 minutes. Then add 10 microliters of 333 millimolar iodoacetamide to the sample and incubate in the dark for 30 minutes at 37 degrees C.Add 10 microliters of trypsin to the mixture and incubate overnight at 37 degrees Celsius.The next day transport the final product to an LC-MS/MS facility or to a third party mass spectrometry lab for analysis. This image shows silver staining of flag epitope tagged ARIP4 expressed in HEK 293 cells after immunal precipitation with a flag specific antibody and separation by electrophoresis. As a control, a mock purification was performed on HEK 293 cells that did not express exogenous proteins.Using our method, it is possible to identify putative nuclear cofactors via their transcriptional factor of interest and further enhance our understanding of transcriptional regulatory mechanisms.

a-protein-preparation-method-for-high-throughput-identification

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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.

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A neuronal lysosome proximity labeling proteomics protocol is described here to characterize the dynamic lysosomal microenvironment in human induced pluripotent stem cell-derived neurons. Lysosomal membrane proteins and proteins that interact with lysosomes (stably or transiently) can be accurately quantified in this method with excellent intracellular spatial resolution in live human neurons.

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Learn a protein preparation method for identifying nuclear cofactor interactions using LC-MS/MS analysis in HEK 293 cells. This immunoprecipitation technique enables high-throughput discovery of transcriptional regulatory protein networks.

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis (Video) | JoVE Journal

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

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