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. Transfeksjon <ol><li> Seed HEK293 celler i 100 mm-kultur plater (2 x 10 6 celler / parabol). Kultur ble cellene i Dulbeccos modifiserte Eagles medium supplementert med 10% føtalt kalveserum, 100 ug / ml streptomycin, og 100 enheter / ml penicillin. Cellene inkuberes i en fuktig inkubator med 5% CO2 og 37 ° C. </li><li> Neste dag, transfektere cellene med 10 mikrogram av FLAG-merket ARIP4 plasmid bruker 40 ul transfeksjon reagens i henhold til produsentens anvisninger 11.</s...

<ol>
	<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>

Effektiv transfeksjon er avgjørende for å oppnå et vellykket resultat med denne protokoll. Derfor anbefaler vi at du bruker Western blot analyse for å fastslå de immunoutfelt FLAG-tagget protein nivåer. Dette trinnet gjør det mulig å verifisere at deres protein av interesse er riktig uttrykt, og dessuten at IP er vellykket gjennomført. FLAG-merket protein nivåer bør også sjekkes før massespektrometri analyse. En mock prøven bør brukes som en negativ kontroll for å unngå å f...

Protein-protein-interaksjoner spiller en viktig rolle i mange biologiske funksjoner. Som sådan, har disse interaksjoner vært innblandet i signaltransduksjon; protein transport over cellemembraner; celle metabolisme; og flere nukleære prosesser, blant annet DNA-replikasjon, DNA-skade reparasjon, rekombinasjon, og transkripsjon 1, 2, 3, 4. Identifisere proteiner som er involvert i disse interaksjonene er derfor avgjørende for å fremme vår forståelse av disse cellulære prosesser. Immunoutfelling (IP) er et validert teknikk som brukes til å analysere protein-protein-interaksjoner. For å lette identifiseringen av ko-immunopresipitert proteiner, blir massespektrometri ofte benyttet 5, 6, 7, 8,9. Ved å målrette et kjent medlem av et proteinkompleks med et antistoff, er det mulig å isolere proteinkomplekset og deretter å identifisere de ukjente komponentene via massespektrometri-analyse. ARIP4 (androgen reseptor-samspill protein 4), en transkripsjons coregulator, samhandler med atom reseptor proteiner for å aktivere eller undertrykke sine mål arrangører i en kontekstavhengig måte 9, 10. For bedre å forstå de transkripsjonsregulerende mekanismer som styrer disse nukleære faktorer, beskriver vi en omfattende fremgangsmåte for å rense og identifisere ARIP4 samspill proteiner ved hjelp av LC-MS / MS-system.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="81">
			<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>
		</tr>
	</tbody>
</table>

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.

Vi identifiserte en sterk ARIP4 signal, rundt 160 kDa, så vel som de av en uekte prøve kontroll og flere andre ukjente proteiner (figur 1). LC-MS / MS-analyse er identifisert både ARIP4 komplekse peptider og de potensielle peptid kofaktorer innenfor brøkdel av FLAG kuler (tabell 1). p62 (Sequestosome1), ble en kjent ARIP4 kofaktor 11 identifisert i analysen (tabell 1), som bekrefter virkningen av dette system ...

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

Vi har etablert en metode for rensing av coregulatory interaksjons proteiner ved anvendelse av LC-MS / MS-system.

En Protein Forberedelse metode for High-throughput identifisering av proteiner samspill med et kjernefysisk kofaktor Bruke LC-MS / MS-analyse

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.

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Metode for å identifisere lavmolekylære inhibitorer av protein-protein samhandling mellom HCN1 og TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Bruke Stillas liposomer for å rekonstituere Lipid-proksimale Protein-protein interaksjoner<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Nuclear Magnetic Resonance spektroskopi for identifisering av flere Phosphorylations med egen Uordnede Proteiner

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Høy gjennomstrømning, absolutt vilje av innholdet i et valgt Protein på vev nivåer ved hjelp av kvantitative Dot Blot analyse (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

En enkel metode for høy gjennomstrømning kjemiske Screening i Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

Effektiv rensing og LC-MS/MS-baserte analysen utvikling for ti-elleve translokasjon-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Tverrgående seksjonering av eldre ris (Oryza sativa L.) Kjerner for skanning av elektronmikroskopiavbildning ved hjelp av pipettespisser som immobiliseringsstøtte

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Fjerning og utskifting av endogene ligander fra Lipidbundne proteiner og allergener

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Et økonomisk og allsidig proteinrensesystem med høy gjennomstrømning ved hjelp av en adapter med flere kolonner

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Prøvepreparering ved hjelp av en Lipid Monolayer-metode for elektronkrystallografiske studier

Electron crystallography is a powerful tool for high-resolution structure determination. Macromolecules such as soluble or membrane proteins can be grown ...