This work was funded by the Wellcome Trust (WT098051).

<ol>
	<li>Goudreault, 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=A+PP2A+phosphatase+high+density+interaction+network+identifies+a+novel+striatin-interacting+phosphatase+and+kinase+complex+linked+to+the+cerebral+cavernous+malformation+3+(CCM3)+protein.">A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein.</a> Mol Cell Proteomics. 8 (1), 157-171 (2009).</li><li>Leitner, 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=Probing+native+protein+structures+by+chemical+cross-linking,+mass+spectrometry,+and+bioinformatics.">Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics.</a> Mol Cell Proteomics. 9 (8), 1634-1649 (2010).</li><li>Rappsilber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+a+beautiful+friendship:+cross-linking/mass+spectrometry+and+modelling+of+proteins+and+multi-protein+complexes.">The beginning of a beautiful friendship: cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes.</a> J Struct Biol. 173 (3), 530-540 (2011).</li><li>Sinz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+cross-linking+and+mass+spectrometry+to+map+three-dimensional+protein+structures+and+protein-protein+interactions.">Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions.</a> Mass Spectrom Rev. 25 (4), 663-682 (2006).</li><li>Walzthoeni, T., Leitner, A., Stengel, F., Aebersold, R. <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+supported+determination+of+protein+complex+structure.">Mass spectrometry supported determination of protein complex structure.</a> Curr Opin Struct Biol. 23 (2), 252-260 (2013).</li><li>Kaake, R. 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=A+new+in+vivo+cross-linking+mass+spectrometry+platform+to+define+protein-protein+interactions+in+living+cells.">A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells.</a> Mol Cell Proteomics. 13 (12), 3533-3543 (2014).</li><li>Kao, 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=Development+of+a+novel+cross-linking+strategy+for+fast+and+accurate+identification+of+cross-linked+peptides+of+protein+complexes.">Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes.</a> Mol Cell Proteomics. 10 (1), (2011).</li><li>Hughes, M. A., Langlais, C., Cain, K., MacFarlane, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterisation+and+reconstitution+of+cell+death+signalling+complexes.">Isolation, characterisation and reconstitution of cell death signalling complexes.</a> Methods. 61 (2), 98-104 (2013).</li><li>Lim, S., Zou, Y., Friedman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+activator+Mirk/Dyrk1B+is+sequestered+by+p38alpha/beta+MAP+kinase.">The transcriptional activator Mirk/Dyrk1B is sequestered by p38alpha/beta MAP kinase.</a> J Biol Chem. 277 (51), 49438-49445 (2002).</li><li>Havugimana, P. C., 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=A+census+of+human+soluble+protein+complexes.">A census of human soluble protein complexes.</a> Cell. 150 (5), 1068-1081 (2012).</li><li>Kirkwood, K. J., Ahmad, Y., Larance, M., 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=Characterization+of+native+protein+complexes+and+protein+isoform+variation+using+size-fractionation-based+quantitative+proteomics.">Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.</a> Mol Cell Proteomics. 12 (12), 3851-3873 (2013).</li><li>Kristensen, A. R., Gsponer, J., Foster, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+approach+for+measuring+temporal+changes+in+the+interactome.">A high-throughput approach for measuring temporal changes in the interactome.</a> Nat Methods. 9 (9), 907-909 (2012).</li><li>Liu, F., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+the+architecture+of+protein+assemblies+and+protein+interaction+networks+by+cross-linking+mass+spectrometry.">Interrogating the architecture of protein assemblies and protein interaction networks by cross-linking mass spectrometry.</a> Curr Opin Struct Biol. 35, 100-108 (2015).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+electrophoretic+techniques+to+identify+protein-protein+interactions.">Native electrophoretic techniques to identify protein-protein interactions.</a> Proteomics. 9 (23), 5214-5223 (2009).</li><li>Heide, H., 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=Complexome+profiling+identifies+TMEM126B+as+a+component+of+the+mitochondrial+complex+I+assembly+complex.">Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.</a> Cell Metab. 16 (4), 538-549 (2012).</li><li>Wessels, H. 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=Analysis+of+953+human+proteins+from+a+mitochondrial+HEK293+fraction+by+complexome+profiling.">Analysis of 953 human proteins from a mitochondrial HEK293 fraction by complexome profiling.</a> PLoS One. 8 (7), 68340 (2013).</li><li>Wessels, H. 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=LC-MS/MS+as+an+alternative+for+SDS-PAGE+in+blue+native+analysis+of+protein+complexes.">LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of protein complexes.</a> Proteomics. 9 (17), 4221-4228 (2009).</li><li>Remmerie, N., 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=Unraveling+tobacco+BY-2+protein+complexes+with+BN+PAGE/LC-MS/MS+and+clustering+methods.">Unraveling tobacco BY-2 protein complexes with BN PAGE/LC-MS/MS and clustering methods.</a> J Proteomics. 74 (8), 1201-1217 (2011).</li><li>Sessler, N., Krug, K., Nordheim, A., Mordmuller, B., Macek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Plasmodium+falciparum+proteasome+using+Blue+Native+PAGE+and+label-free+quantitative+mass+spectrometry.">Analysis of the Plasmodium falciparum proteasome using Blue Native PAGE and label-free quantitative mass spectrometry.</a> Amino Acids. 43 (3), 1119-1129 (2012).</li><li>Bode, D., Yu, L., Tate, P., Pardo, M., Choudhary, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Two+Distinct+Nucleosome+Remodeling+and+Deacetylase+(NuRD)+Complex+Assemblies+in+Embryonic+Stem+Cells.">Characterization of Two Distinct Nucleosome Remodeling and Deacetylase (NuRD) Complex Assemblies in Embryonic Stem Cells.</a> Mol Cell Proteomics. 15 (3), 878-891 (2016).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MaxQuant+enables+high+peptide+identification+rates,+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification.">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nat Biotechnol. 26 (12), 1367-1372 (2008).</li><li>Cox, 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=A+practical+guide+to+the+MaxQuant+computational+platform+for+SILAC-based+quantitative+proteomics.">A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.</a> Nat Protoc. 4 (5), 698-705 (2009).</li><li>Kim, J. 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=A+novel+regulatory+factor+recruits+the+nucleosome+remodeling+complex+to+wingless+integrated+(Wnt)+signaling+gene+promoters+in+mouse+embryonic+stem+cells.">A novel regulatory factor recruits the nucleosome remodeling complex to wingless integrated (Wnt) signaling gene promoters in mouse embryonic stem cells.</a> J Biol Chem. 287 (49), 41103-41117 (2012).</li><li>Lauberth, S. M., Rauchman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+12-amino+acid+motif+in+Sall1+recruits+the+nucleosome+remodeling+and+deacetylase+corepressor+complex.">A conserved 12-amino acid motif in Sall1 recruits the nucleosome remodeling and deacetylase corepressor complex.</a> J Biol Chem. 281 (33), 23922-23931 (2006).</li><li>Miller, 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=Sall4+controls+differentiation+of+pluripotent+cells+independently+of+the+Nucleosome+Remodelling+and+Deacetylation+(NuRD)+complex.">Sall4 controls differentiation of pluripotent cells independently of the Nucleosome Remodelling and Deacetylation (NuRD) complex.</a> Development. 143 (17), 3074-3084 (2016).</li><li>Dharmarajan, V., Lee, J. H., Patel, A., Skalnik, D. G., Cosgrove, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+WDR5+interaction+(Win)+motif+recognition+in+human+SET1+family+histone+methyltransferases.">Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases.</a> J Biol Chem. 287 (33), 27275-27289 (2012).</li><li>Schwanhausser, B., 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=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7347), 337-342 (2011).</li><li>Smits, A. H., Jansen, P. W., Poser, I., Hyman, A. A., Vermeulen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+chromatin-associated+protein+complexes+revealed+by+label-free+quantitative+mass+spectrometry-based+proteomics.">Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.</a> Nucleic Acids Res. 41 (1), 28 (2013).</li><li>Lambert, J. P., Tucholska, M., Pawson, T., Gingras, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+DNA+shearing+in+standard+affinity+purification+allows+simultaneous+identification+of+both+soluble+and+chromatin-bound+interaction+partners.">Incorporating DNA shearing in standard affinity purification allows simultaneous identification of both soluble and chromatin-bound interaction partners.</a> J Proteomics. 100, 55-59 (2014).</li><li>Wittig, I., Braun, H. P., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1 (1), 418-428 (2006).</li><li>Schagger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis.">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal Biochem. 217 (2), 220-230 (1994).</li><li>Schagger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form.">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Anal Biochem. 199 (2), 223-231 (1991).</li><li>Camacho-Carvajal, M. M., Wollscheid, B., Aebersold, R., Steimle, V., Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+Blue+native/SDS+gel+electrophoresis+of+multi-protein+complexes+from+whole+cellular+lysates:+a+proteomics+approach.">Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.</a> Mol Cell Proteomics. 3 (2), 176-182 (2004).</li><li>Liu, F., Rijkers, D. T., Post, H., Heck, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+profiling+of+protein+assemblies+by+cross-linking+mass+spectrometry.">Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry.</a> Nat Methods. 12 (12), 1179-1184 (2015).</li></ol>

The authors have nothing to disclose.

Burada, protein kompleksleri çözmek için niceliksel kütle spektrometrisi ile kombinasyon halinde mavi ile doğal jel elektroforezi, ardından afinite saflaştırma kullanılmasını tarif eder. Bu yaklaşım, fonksiyonel protein takımlar halinde tek boyutlu protein etkileşim listeleri aydınlatmak için bir yöntem sunmaktadır. Bu epitop etiketli proteinlerin kullanımına dayalı bir yöntemi göstermektedir. etiketli proteini eksprese eden bir hücre çizgisi mevcut değildir, ancak alternatif, ilgi konusu proteine ​​karşı antikorlar kullanmak için, doğal rekabet elüsyon elde etmek için uygun bir peptit olması koşuluyla olabilir. Başlatıcı madde ve boncuk miktarı miktarı, hedef proteinin ekspresyon düzeyi ile ilgili olarak modifiye edilmesi gerekebilir. Normal olarak, başlangıç malzemesi 2-5 8 x 10 hücreleri ile bu protokolü gerçekleştirmek ve bu daha düşük bir ifade seviyesine sahip proteinler için yeterlidir. liziz tamponu bileşimi elde etmek için ampirik olarak seçilmelidiryakın yem çözünürleşmesini tamamlayın. Bu bağlanma, özellikle kromatin veya zar proteinleri, proteinlerin bazı türleri için zor olabilir. Alternatif seçenekler çalışılan protein kompleksi, yüksek tuz veya kromatin bağlayıcı proteinler, 20, 29 için sonikasyon ve / veya nükleaz işlemi kullanılarak stabil olması koşuluyla, tuz miktarı artan içerir. Membran proteinleri durumunda, DD veya digitonin deterjan geçiş 30 tavsiye edilebilir. DNA bağlama proteinleri durumunda, saflaştırma aşaması 20 esnasında benzonaz gibi bir nükleaz dahil etmek yararlıdır. Nükleik asitlerin tamamen çıkarılması tespit etkileşimleri proteinler arasında oluştuğu ve DNA tarafından aracılık olup olmadığını kontrol eder. Bu yaklaşımı kullanarak araştırma altındaki kompleksin kararlılığı olduğunda kritik bir unsur dikkate. usul, çok uzun ve preservin kapsayabilirg protein kompleksi gece boyunca karıştırılmıştır. İki kromatin yeniden modelleme kompleksleri (D. Bode ve M. Pardo, veriler gösterilmemiştir) ile iyi bir başarı elde etmiş, fakat bu değerlendirilmelidir. Gerekirse burada afinite arıtma aşaması kısalabilir. Bu tür, boyut dışlama kromatografisi gibi doğal fraksiyonasyon izin alternatif teknikler, geniş bir protein kompleksleri karakterize etmek için 50 yıldan fazla bir süredir kullanılmaktadır. Bu, diğerleri mavi doğal PAGE çözünürlüğü boyut dışlama kromatografisi 20, 31, 32, 33 kullanılarak elde edilene göre daha üstün olduğunu göstermiştir. mavi doğal PAGE bir diğer avantajı da pahalıdır kromatografi sistemleri, gerektirmediğini, ancak yerine laboratuvarlarda yaygın protein elektroforetik ekipman kullanır. açısından uygulamalı zaman, bu yöntem geleneksel GELC-MS / MS a daha çok işim içermezpproach veya çevrimdışı kromatografik ayırma. En fraksiyonasyon teknikleri gibi Ancak, onların çözümüne bir sınırı vardır. çok homojen veya kütle ve şekil olarak yakın Kompleksleri mavi doğal PAGE tarafından sunulan çözünürlük ve paylaşılan alt birimleri ile ayrı kompleksleri çözümünde evrensel başarılı olmayabilir aşağıda bildirildiği gibi dolayısıyla protokol ötesinde olabilir. Umut verici bir şekilde, üç alt-birimleri (M Pardo hazırlama yazısı) paylaşan iki çok benzer tetramerik komplekslerin ayrılması başarılı olmuştur. kütle spektrometresi giderek duyarlı hale gelmiştir beri spesifik olmayan interactors ve kirletici maddelerin dahi dakika miktarları AP-MS örneklerinde tespit edilebilir. Burada sunulan bir yaklaşım daha yüksek moleküler ağırlıkta yem taşıma zirve noktaları ile çakışmaktadır göç pikleri ile protein almaya odaklanarak afinite saflaştırma arka plan kirlenme gerçek interactors ayrımı da yardımcı olabilirmonomerik protein. Birkaç grup, önceki izolasyon 10, 11, 12, 34 ihtiyaç olmaksızın hücresel ölçekte protein kompleksleri tanımlamak için, protein korelasyon profil ardından ayırma tekniği kullanılmıştır. Ancak, bu alt stokiyometrik etkileşimlerini tespit etmek için başarısızlıkla sonuçlanabilir. afinite saflaştırma ile bir zenginleştirme aşamasının dahil edilmesi, bu üstesinden gelmeye yardımcı olabilir. Burada tarif edilen bir yaklaşım, belirli bir protein, aynı hücre kapsamında yer alır birden fazla kompleks protein kompleksleri topoloji keşfetmek ve çözülmesi için genel olarak kullanışlı olmalıdır. strateji, protein kompleksleri gidermek için pahalı kromatografik parçalanma ekipmana sahip olmayabilir laboratuvarlara basit ve elverişlidir.

hücrelerde, pek çok protein geçici protein-protein etkileşimleri yoluyla veya stabil protein düzeneklerinin oluşturulması suretiyle işlevleri yerine getirir. Protein etkileşimleri belirlemek tamamen anlaşılması hücresel süreçleri için çok önemlidir. kütle spektrometrisi (AP-MS) ile kombinasyon halinde afinite saflaştırma doğal protein etkileşimlerini tespit etmek için en yaygın olarak kullanılan yöntemlerden biridir. Son on yılda elde enstrüman yetenekleri önemli gelişmeler bu yaklaşımın son derece güçlü hale getirmiştir. AP-MS deneyler tarafından tespit etkileşimleri yem ve onlardan beslenen arasındaki doğrudan ve dolaylı dernekler bir karışımını içermektedir dikkat etmek önemlidir. Buna ek olarak, genellikle proteinler farklı biyolojik rollere sahip olabilir aynı hücresel kapsamında çeşitli kompleksler yer alır ve bu nedenle, AP-MS ile tespit edilir uygulayıcıların farklı protein montajlar veya işlevsel birimlerin bir karışımı temsil edebilir. Türetmek mümkün değildirörneğin topolojik bilgi, basit AP-MS deneyi ile oluşturulan proteinlerin mono- boyutlu listelerden önsel. Ancak, bu teknik, bu derlemeler gidermek için, bir ya da daha fazla yöntem ile birleştirerek protein kompleksleri mimarisini tanımlamak için daha fazla kullanılabilir. AP-MS yoluyla tespit protein etkileşimleri topoloji çözmek amacıyla, çeşitli stratejiler uygulanmıştır. Bir yaklaşım, yemler 1 ile deneyler önceki turunda tanımlanan avlar iteratif AP-MS deneyler sağlamaktır. çok bilgilendirici olsa da, bu hem deneysel hem de analitik oldukça emek yoğun bir iştir. Kütle spektrometrisi ile kombinasyon halinde protein çapraz bağlanma artan protein kompleksleri, 2, 3, 4, 5 topolojik bilgi elde etmek için kullanılıyor. Ancak, computatioçapraz bağlı peptitlerin nihai analiz yine zor bir görev kalır ve bu nedenle, iş akışında darboğazdır. MS-yarılabilir çapraz-bağlama maddeleri gelişi proteinleri 6, 7 etkileşimde yakın amino asit artıklarının eşleme kolaylaştırmalıdır. Başka bir alternatif önce ortogonal ayırma teknikleri 8, 9 ile afinite saflaştırmasını birleştirmektir. Jel filtrasyonu ya da iyon değişimi ya da sukroz gradyanlı fraksiyonasyonu ile kromatografik ayırma yakın zamanda by-pass karmaşık izolasyon aşaması 10, 11, 12, 13, bir sistem genelinde düzeyde multiprotein komplekslerini tarif kantitatif kütle spektrometrisi ile kombinasyon halinde kullanılmıştır. Mavi doğal poliakrilamid jel elektroforezi (BN-PAGE), yaygın olarak uygulanmıştırdoğal protein etkileşimleri, mitokondriyal membran protein kompleksleri 14 kapsayan, genel olarak bu araştırmak. Bu ayırma tekniği de son zamanlarda mitokondriyal kompleksleri 15 üzerinde sadece etiket serbest protein miktarının ve korelasyon profili ile kombinasyon halinde kullanılan 16, 17, aynı zamanda, 19, bütün hücreler 18 diğer protein kompleksleri çözülmesi için. Biz daha sonra doğal fraksiyonasyon yaklaşımları ve kantitatif MS ile afinite saflaştırma kombinasyonu belirli bir protein ihtiva eden birçok protein tertibatları çözmek için yararlı bir strateji sağlamalıdır varsayıldı. Burada kantitatif kütle sp ardından izole komplekslerinin mavi doğal poliakrilamid jel elektroforezi ile genel epitop bazlı afinite saflaştırmasını eden bir yöntem, tarifectrometry ve protein korelasyon profil, bir protein dahil olabilir birden çok derleme çözmek için. Bu, bir epitop ucuna birleştirilen bir ilgi, bir protein, fizyolojik bolluk yakın elde etmek ve etkili bir yerli sağlamak için endojen mahalden türetilmektedir ifade edilir, fare embriyonik kök hücreleri kullanır karmaşık izolasyonu. Bu yaklaşım, bir protein, geleneksel GELC-MS 20 den daha fazla çalışma gerektiren iken, aralarındaki korelasyon profillerine göre farklı takımlar halinde çözdürerek, yürütmektedir birden çok etkileşimi unravels.

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Protein G Dynabeads</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">10003D</td>
			<td style="width:452px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">FLAG antibody M2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Tween-20, protein grade, 10% solution</td>
			<td style="width:285px;">Millipore</td>
			<td style="width:220px;">655206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Dimethyl pimelimidate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">80490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">0.2 M Triethanolamine buffer, pH 8.2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">T0449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">3x FLAG peptide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Vivaspin 5K NMWCO, PES</td>
			<td style="width:285px;">Sartorius</td>
			<td style="width:220px;">VS0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativePAGE 3-12% Bis-Tris gel</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Running Buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Cathode Additive</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">4x NativePAGE sample loading buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativeMARK</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">LC0725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NativePAGE 5% G-250 sample additive&#160;</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc conical bottom 96-well plate, polypropylene</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">249944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Multiscreen HTS 96-well filtration system</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">MSDVN6550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc 96-well cap natural</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">276002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Iodoacetamide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">I1149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Acetonitrile, HPLC grade</td>
			<td style="width:285px;">Fisher Scientific</td>
			<td style="width:220px;">A/0627/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Trypsin, sequencing grade</td>
			<td style="width:285px;">Roche</td>
			<td style="width:220px;">11418475001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Software</td>
			<td style="width:285px;"></td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MaxQuant (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111795/maxquant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Perseus (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111810/perseus</td>
		</tr>
	</tbody>
</table>

resolving affinity purified protein complexes by blue native page and protein correlation profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Kütle Spektrometresi Afinite arıtma Mavi doğal protein Korelasyon profil iş akışı (ABC-MS), bir strateji, Şekil 7&#39;de tasvir edilmiştir. ilgi konusu bir proteinin yaklaşık yerel protein kompleksleri (bu durumda FLAG) bir epitop etiketine karşı antikorlar kullanılarak afinite saflaştırma ve rekabetçi elüsyon ile izole edilir. kompleksleri BN-PAGE ile çözülür ve bütün jel şeridi 48 bölüme eksize edilir, ve av tüfeği LC-MS / MS için hazırlanmıştır. Kantitatif MS bilgileri mavi doğal ayırma boyunca tespit edilen her bir protein için bir geçiş profili oluşturmak için kullanılır. etkileşen proteinler, üst üste zirveleri ile bir tat karmaşık görüntüleme içindeki göç profillerini oluşturulur. ilgi konusu bir proteinin birden fazla montaj yer alır, birden fazla tepe noktası, kendi göç profilinde görülmektedir alt kompleksleri mavi doğal jel çözme gücü dahilindedir verilmiştir. mig sistematik karşılaştırılmasırasyon profilleri hiyerarşik kümeleme kullanarak profilleme protein korelasyon elde edilebilir. Dendrogramını ve fraksiyonlar için tepe yoğunlukları farklı protein kompleksleri ait proteinler (Şekil 8) etkileşimde tanımlanmasını kolaylaştıran bir ısıyla harita içinde görülür. Biz Mta2, kompleks 20 yeniden modelleme nurd kromatin bir çekirdek alt biriminin etkileşim ortakları analiz etmek için bu stratejiyi kullandı. Şekil 9&#39;da gösterildiği gibi, Mta2 göç profili yüksek bolluk gösteren aşağıdaki kütle tepe, 700 kDa ve 1.2 MDA arasındaki belirgin yoğunluklarının iki tepe sergiledi. MTA1 / 3, HDAC1 / 2 ve Mbd3 dahil olmak üzere diğer nurd çekirdek alt birimleri, alt birimler, yani Chd4 bazı piklerin olsa aynı ayırma paternini b Gatad2a ve / Rbbp4 / 7, (Şekil 9A ters bolluk dağılım B). Buna karşılık, Cdk profili2ap1 Wnt gen promoteri 23 nurd kompleks düzelir bir düzenleyici faktör, sadece yüksek kütle zirve (Şekil 9C) görüntülenir ve böylece Sall4 da nurd 20, 24, 25 bağlandığı gösterilmiş olan bir transkripsiyon represör yaptı. Bu nedenle, mavi doğal PAGE ile afinite saflaştırılmış Mta2 ilişkili proteinlerin parçalanması nurd kompleksinin iki farklı şeklini çözmek mümkün oldu. ABC-MS de özel protein kişiler bunları atanmaktadır yeni interactors tanımlanmasına izin verir. Bir ısı harita temsil proteinler kümeleri ve fraksiyon yoğunluklarının incelenmesi sayesinde iki nurd p ile çakışan iki göç zirveleri gösteren nurd alt birimleri ve Wdr5 ile Wdr5, MLL metiltransferaz kompleksi 26 bir düzenleyici alt birimi, arasında güçlü bir ilişki tespitWdr5 ve nurd arasında yeni bir etkileşim olduğunu eaks (Şekil 8). Bu, boyut dışlama kromatografisi 20 eş-immünopresipitasyon ve ikili göç ile bu etkileşimi doğruladı. Hiyerarşik kümeleme mesafe metrik hesaplaması seçimi kümeleri ve dolayısıyla korelasyon şeklini etkileyecektir. Biz proteinin veya kompleksin mevcut etkileşim bilgisi ile en iyi uyum elde etmek için farklı ölçümlerin denemeler yapmanızı öneririz. Genellikle Manhattan (L1) mesafe metriği 20 ile en iyi sonuçlar elde edilmiştir. Pearson korelasyon veya bir Öklid mesafe ölçümlerini alternatif ayırma teknikleri 10, 11, 12 göre kompleksler belirlenmesi için rapor edilmiştir. quanMavi doğal fraksiyonlardan elde edilen titative MS verileri, aynı zamanda protein kompleksleri stokiyometri belirlemek için kullanılabilir. IBAQ (yoğunluk göre mutlak miktar) değeri olarak belirlenen proteinler 27, 28 nispi bolluk bir ölçüsünü sağlar. bir geçiş profili tepe içindeki tüm fraksiyonlarda protein kompleksi üyeleri için iBAQ değerleri göreceli miktarlarını elde etmek için yem proteinin normalize edilir. Belirli bir protein kompleksi üyesi için bir profil zirve boyunca normalize iBAQs yatay bir seyir takip etmeli ve trend değerleri yem proteine ​​göre etkileşim proteinlerin stokiyometrisini yansıtmaktadır. Stokiyometri hesaplamalar daha ayrıntılı bir gösterimi için, 20 bkz. <img alt="Şekil 7," src="/files/ftp_upload/55498/55498fig7.jpg" /> Şekil 7: Şematik akışı özetleyen ABC-MS strategy. Bu rakam 20 ile modifiye edilir. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig7large.jpg" target="_blank">Bu rakamın büyük halini görmek için buraya tıklayın.</a> <img alt="Şekil 8," src="/files/ftp_upload/55498/55498fig8.jpg" /> Mta2 interactors BN-PAGE migrasyon profilleri Şekil 8. Hiyerarşik kümeleme. Mta2 ilişkili proteinler göç profilleri benzerliğine göre kümelenmiş. Sadece nurd kompleksi içeren ısı-haritasının bir alt (mavi bir kutu içine) gösterilmektedir. Sarı kutu nurd kompleksi ile Wdr5 güçlü bir korelasyon vurgulamaktadır. açıklamalı moleküler ağırlıklar aynı jel içinde Protein standartlarının geçiş mesafeleri tahmin edilmiştir. Bu araştırma, başlangıçta Biyokimya ve Mo için 20 Amerikan Derneği Moleküler ve Hücresel Proteomics yayımlandı<html> lecular Biyoloji. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig8large.jpg" target="_blank">Bu rakamın büyük halini görmek için buraya tıklayın.</a> <img alt="Şekil 9," src="/files/ftp_upload/55498/55498fig9.jpg" /> Nurd alt birimleri ve ilişkili proteinlerin Şekil 9. BN-PAGE göç profili. A) benzer yoğunlukta bir model mevcut Mta2 ve nurd alt birimi. B) ters yoğunluk desenli Mta2 ve nurd alt birimi. Sadece yüksek molekül ağırlıklı nurd bir oluşum halinde, C) Mta2 ve Cdk2ap1. açıklamalı moleküler ağırlıklar Protein standartlarının göç profili tahmin edildi. Bu araştırma, başlangıçta Moleküler ve Hücresel Proteomics 20 Biyokimya ve Moleküler Biyoloji American Society yayımlandı.&quot;_blank&gt; bu rakamın büyük halini görmek için lütfen buraya tıklayın. <img alt="Ek Dosya" src="/files/ftp_upload/55498/proteinGroupsDB04.jpg" /> Ek veriler: Örnek veri kümesi. bir ABC-MS deneyinde protein tanımlamalar miktar değerleri içeren proteingroups.txt dosyasını MaxQuant türevi. Bu araştırma, başlangıçta Moleküler ve Hücresel Proteomics 20 Biyokimya ve Moleküler Biyoloji American Society yayımlandı. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/proteinGroupsDB04.txt" target="_blank">Bu dosyayı indirmek için buraya tıklayın.</a>

Watch this Scientific Journal Video about Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling at JoVE.com

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Burada bir etiket bilgilerini kantitatif kütle spektrometresi kullanılarak protein korelasyon profil ardından afinite protein komplekslerinin saflaştırılması ve mavi doğal PAGE ile onların ayrılması için protokoller, mevcut. Bu yöntem, farklı protein kompleksleri halinde interactomes çözmek için yararlıdır.

Mavi Yerli PAGE ve Protein İlişkisi Profil Yöntemi ile Affinity Saf protein Kompleksler çözümleniyor

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

resolving-affinity-purified-protein-complexes-blue-native-page

Nanyang Technological University

Research

JoVE Journal

Biochemistry

13.7K Görüntüleme.  Wellcome Trust Sanger Institute.  Burada bir etiket bilgilerini kantitatif kütle spektrometresi kullanılarak protein korelasyon profil ardından afinite protein komplekslerinin saflaştırılması ve mavi doğal PAGE ile onların ayrılması için protokoller, mevcut. Bu yöntem, farklı protein kompleksleri halinde interactomes çözmek için yararlıdır. 

Video: Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled memeli hücrelerinden protein kompleksi olan ele geçirme kapasitesini

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Biyokimya

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

Ökaryotik Hücrelerde Protein Kompleksleri Tandem Affinity Arıtma

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

Multimer-PAGE: Biyolojik örnekler olarak yakalamak için bir yöntem ve özümleme protein kompleksleri

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

Protein-RNA Komplekslerinin Geliştirilmiş Algılaması için Yatay Jel Elektroforezi

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

Split-BioID — Proteomik analiz onların yerel hücresel ortamında içeriğe özel Protein kompleksleri

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 in 1: tek adımlı benzeşme arıtma paralel analizi için Protein-Protein ve Protein-metaboliti kompleksleri

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Thylakoid membran Protein kompleksleri tarafından mavi yerli jel elektroforez Analizi

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Kloroplast Translocon Protein kompleksleri dokunun etiketini kullanarak benzeşme arıtma

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Transkripsiyon faktörü-DNA bağlayıcı Affinities floresans mikroskobu kullanarak rekabet titrasyon tarafından yüksek hassasiyet ölçümü

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Protein katlanması, taşıma ve canlı hücreler radyoaktif darbe Chase tarafından bozulması Analizi

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...