Name: a protein preparation method for the high-throughput identification of proteins interacting with a nuclear cofactor using lc-ms/ms analysis
Uploaded: 2017-01-24T12:30:00
Description: Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

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. trasfezione <ol><li> Seed HEK293 cellule in piastre di coltura da 100 mm (2 x 10 6 cellule / piatto). Cultura le cellule in mezzo di Eagle modificato di Dulbecco supplementato con 10% di siero fetale di vitello, 100 ug / ml di streptomicina e 100 unità / ml di penicillina. Incubare le cellule in un incubatore umidificato al 5% di CO 2 e 37 ° C. </li><li> Il giorno successivo, trasfezione le cellule con 10 mg di FLAG-tagged ARIP4 plasmide con 40 ml di reagente di trasfezione secondo le istruz...

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	<li>De Las Rivas, J., Fontanillo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions+essentials:+key+concepts+to+building+and+analyzing+interactome+networks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+interactions+involved+in+cellular+signaling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+PLIN+proteins,+ATGL+and+CGI-58,+interactions+at+rest+and+following+stimulated+contraction.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactive-state+preassembly+of+G(q)-coupled+receptors+and+G(q)+heterotrimers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+with+chromatin+modifiers+that+occupies+E2F-+and+Myc-responsive+genes+in+G0+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+histone+modifications+mediated+by+a+CtBP+co-repressor+complex.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+human+papillomavirus+type+16+E7+oncoprotein+with+the+600-kDa+retinoblastoma+protein-associated+factor,+p600.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+identification+of+Akt+interacting+proteins+by+two-step+chemical+crosslinking,+co-immunoprecipitation+and+mass+spectrometry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry-based+immuno-precipitation+proteomics+-+the+user's+guide.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Suppression+by+Transient+Recruitment+of+ARIP4+to+Sumoylated+nuclear+receptor+Ad4BP/SF-1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+autophagic+receptor+p62+regulates+the+abundance+of+transcriptional+coregulator+ARIP4+during+nutrient+starvation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+an+intracellular+lectin,+calnexin,+from+Aspergillus+oryzae+using+N-glycan-conjugated+beads.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dyrk1A+potentiates+steroid+hormone-induced+transcription+via+the+chromatin+remodeling+factor+Arip4.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+immunoprecipitation+mass+spectrometry+of+endogenous+proteins+(RIME)+for+analysis+of+chromatin+complexes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+applied+to+proteomic+analysis.">Capillary electrophoresis applied to proteomic analysis.</a> J.Sep.Sci. 32 (8), 1175-1188 (2009).</li></ol>

trasfezione efficiente è essenziale per ottenere un risultato di successo con questo protocollo. Di conseguenza, si consiglia di utilizzare analisi Western Blot per determinare i livelli di proteina FLAG-tagged immunoprecipitati. Questa fase permette di verificare che la proteina di interesse è correttamente sovraespresso e, inoltre, che il PI è stato eseguito con successo. livelli di proteina FLAG-tag dovrebbero essere controllati prima della spettrometria di massa. Un campione finta dov...

Le interazioni proteina-proteina svolgono un ruolo importante in molte funzioni biologiche. In quanto tali, queste interazioni sono stati implicati nella trasduzione del segnale; proteine ​​di trasporto attraverso le membrane cellulari; metabolismo cellulare; e diversi processi nucleari, tra cui la replicazione del DNA, il DNA di riparazione danni, ricombinazione, e trascrizione 1, 2, 3, 4. Identificare le proteine ​​coinvolte in queste interazioni è quindi fondamentale per far progredire la nostra comprensione di questi processi cellulari. Immunoprecipitazione (IP) è una tecnica validata utilizzato per analizzare le interazioni proteina-proteina. Per facilitare l&#39;identificazione di proteine co-immunoprecipitazione, spettrometria di massa è spesso utilizzato 5, 6, 7, 8,9. Prendendo di mira un membro noto di un complesso proteico con un anticorpo, è possibile isolare il complesso proteico e per identificare successivamente i suoi componenti sconosciuti tramite spettrometria di massa. ARIP4 (recettore androgeno-interagendo proteina 4), un coregulator trascrizionale, interagisce con le proteine dei recettori nucleari, al fine di attivare o reprimere i suoi promotori bersaglio in un modo dipendente dal contesto 9, 10. Per comprendere meglio i meccanismi regolatori trascrizionali che regolano questi fattori nucleari, si descrive un metodo completo per purificare ed identificare ARIP4 proteine ​​che interagiscono con il sistema LC-MS / MS.

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			<td height="81" style="height:81px;width:216px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:71px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11668019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Protease Inhibitor Cocktail (EDTA free) (100x)</td>
			<td style="width:71px;">nacalai tesque</td>
			<td style="width:119px;">03969-21</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">ANTI-FLAG M2 Affinity Gel</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2220</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Micro Bio-Spin Columns</td>
			<td style="width:71px;">BIO-RAD</td>
			<td style="width:119px;">732-6204</td>
			<td></td>
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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.

Abbiamo identificato un segnale forte ARIP4, circa 160 KDa, così come quelli di un controllo di esempio finto e diverse altre proteine sconosciute (Figura 1). Analisi LC-MS / MS identificato entrambi i peptidi complessi ARIP4 ei potenziali cofattori peptide all&#39;interno della frazione di perline FLAG (Tabella 1). p62 (Sequestosome1), un noto cofattore ARIP4 11 è stato identificato nell&#39;analisi (Tabella 1),</strong...

Watch this Scientific Journal Video about A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis at JoVE.com

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

Abbiamo stabilito un metodo per la purificazione di proteine ​​di interazione coregolamentazione utilizzando il sistema LC-MS / MS.

Una proteina metodo di preparazione per l&#39;high-throughput identificazione di proteine ​​che interagiscono con un cofattore nucleare Utilizzando LC-MS / MS Analysis

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.

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