Work in the Ait-Si-Ali lab was supported by the Association Fran&#231;aise contre les Myopathies T&#233;l&#233;thon (AFM-T&#233;l&#233;thon); Institut National du Cancer (INCa); Agence Nationale de la Recherche (ANR), Fondation Association pour la Recherche sur le Cancer (Fondation ARC); Groupement des Entreprises Fran&#231;aises pour la Lutte contre le Cancer (GEFLUC); CNRS; Universit&#233; Paris Diderot and the ''Who Am I?'' Laboratory of Excellence #ANR-11- LABX-0071 funded by the French Government through its ''Investments for the Future'' program operated by the ANR under grant #ANR-11-IDEX-0005-01. EB was supported by an INCa grant.

<ol>
	<li>Ait-Si-Ali, S., 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+Suv39h-dependent+mechanism+for+silencing+S-phase+genes+in+differentiating+but+not+in+cycling+cells.">A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells.</a> Embo J. 23 (3), 605-615 (2004).</li><li>Buckingham, M. <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+development+and+the+role+of+the+myogenic+regulatory+factors.">Skeletal muscle development and the role of the myogenic regulatory factors.</a> Biochem Soc Trans. 24 (2), 506-509 (1996).</li><li>Arnold, H. H., Winter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+differentiation:+more+complexity+to+the+network+of+myogenic+regulators.">Muscle differentiation: more complexity to the network of myogenic regulators.</a> Curr Opin Genet Dev. 8 (5), 539-544 (1998).</li><li>Buckingham, M. <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+formation+in+vertebrates.">Skeletal muscle formation in vertebrates.</a> Curr Opin Genet Dev. 11 (4), 440-448 (2001).</li><li>Yun, K., Wold, B. <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+determination+and+differentiation:+story+of+a+core+regulatory+network+and+its+context.">Skeletal muscle determination and differentiation: story of a core regulatory network and its context.</a> Curr Opin Cell Biol. 8 (6), 877-889 (1996).</li><li>Molkentin, J. D., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+regulatory+networks+for+muscle+development.">Defining the regulatory networks for muscle development.</a> Curr Opin Genet Dev. 6 (4), 445-453 (1996).</li><li>Arnold, H. H., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+inactivation+of+myogenic+factor+genes+reveals+their+role+during+mouse+myogenesis:+a+review.">Targeted inactivation of myogenic factor genes reveals their role during mouse myogenesis: a review.</a> Int J Dev Biol. 40 (1), 345-353 (1996).</li><li>Puri, P. L., Sartorelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+muscle+regulatory+factors+by+DNA-binding,+interacting+proteins,+and+post-transcriptional+modifications.">Regulation of muscle regulatory factors by DNA-binding, interacting proteins, and post-transcriptional modifications.</a> J Cell Physiol. 185 (2), 155-173 (2000).</li><li>Tapscott, 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=The+circuitry+of+a+master+switch:+Myod+and+the+regulation+of+skeletal+muscle+gene+transcription.">The circuitry of a master switch: Myod and the regulation of skeletal muscle gene transcription.</a> Development. 132 (12), 2685-2695 (2005).</li><li>Lassar, A. B., Paterson, B. M., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+of+a+DNA+locus+that+mediates+the+conversion+of+10T1/2+fibroblasts+to+myoblasts.">Transfection of a DNA locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts.</a> Cell. 47 (5), 649-656 (1986).</li><li>Davis, R. L., Weintraub, H., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+single+transfected+cDNA+converts+fibroblasts+to+myoblasts.">Expression of a single transfected cDNA converts fibroblasts to myoblasts.</a> Cell. 51, 987-1000 (1987).</li><li>Tapscott, S. 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=MyoD1:+a+nuclear+phosphoprotein+requiring+a+myc+homology+region+to+convert+fibroblasts+to+myoblasts.">MyoD1: a nuclear phosphoprotein requiring a myc homology region to convert fibroblasts to myoblasts.</a> Science. 242, 405-411 (1988).</li><li>Weintraub, 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=Activation+of+muscle-specific+genes+in+pigment,+nerve,+fat,+liver,+and+fibroblast+cell+lines+by+forced+expression+of+MyoD.">Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.</a> Proc Natl Acad Sci U S A. 86 (14), 5434-5438 (1989).</li><li>Mal, A., Harter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoD+is+functionally+linked+to+the+silencing+of+a+muscle-specific+regulatory+gene+prior+to+skeletal+myogenesis.">MyoD is functionally linked to the silencing of a muscle-specific regulatory gene prior to skeletal myogenesis.</a> Proc Natl Acad Sci U S A. 100 (4), 1735-1739 (2003).</li><li>Ohkawa, Y., Marfella, C. G., Imbalzano, A. N. <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+specification+by+myogenin+and+Mef2D+via+the+SWI/SNF+ATPase+Brg1.">Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1.</a> Embo J. 25 (3), 490-501 (2006).</li><li>Ling, B. 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=Lysine+methyltransferase+G9a+methylates+the+transcription+factor+MyoD+and+regulates+skeletal+muscle+differentiation.">Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation.</a> Proc Natl Acad Sci U S A. 109 (3), 841-846 (2012).</li><li>Zhang, C. L., McKinsey, T. A., Olson, E. N. <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+class+II+histone+deacetylases+with+heterochromatin+protein+1:+potential+role+for+histone+methylation+in+control+of+muscle+differentiation.">Association of class II histone deacetylases with heterochromatin protein 1: potential role for histone methylation in control of muscle differentiation.</a> Mol Cell Biol. 22 (20), 7302-7312 (2002).</li><li>Forcales, S. V., 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=Signal-dependent+incorporation+of+MyoD-BAF60c+into+Brg1-based+SWI/SNF+chromatin-remodelling+complex.">Signal-dependent incorporation of MyoD-BAF60c into Brg1-based SWI/SNF chromatin-remodelling complex.</a> The EMBO journal. 31 (2), 301-316 (2011).</li><li>Nakatani, Y., Ogryzko, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoaffinity+purification+of+mammalian+protein+complexes.">Immunoaffinity purification of mammalian protein complexes.</a> Methods Enzymol. 370, 430-444 (2003).</li><li>Ouararhni, K., 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=The+histone+variant+mH2A1.1+interferes+with+transcription+by+down-regulating+PARP-1+enzymatic+activity.">The histone variant mH2A1.1 interferes with transcription by down-regulating PARP-1 enzymatic activity.</a> Genes Dev. 20 (23), 3324-3336 (2006).</li><li>Fritsch, L., 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+subset+of+the+histone+H3+lysine+9+methyltransferases+Suv39h1,+G9a,+GLP,+and+SETDB1+participate+in+a+multimeric+complex.">A subset of the histone H3 lysine 9 methyltransferases Suv39h1, G9a, GLP, and SETDB1 participate in a multimeric complex.</a> Mol Cell. 37 (1), 46-56 (2010).</li><li>Robin, P., Fritsch, L., Philipot, O., Svinarchuk, F., Ait-Si-Ali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modifications+of+histones+H3+and+H4+associated+with+the+histone+methyltransferases+Suv39h1+and+G9a.">Post-translational modifications of histones H3 and H4 associated with the histone methyltransferases Suv39h1 and G9a.</a> Genome Biol. 8 (12), R270 (2007).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat Protoc. 4 (1), 44-57 (2009).</li><li>Huang da, W., Sherman, B. T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatics+enrichment+tools:+paths+toward+the+comprehensive+functional+analysis+of+large+gene+lists.">Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.</a> Nucleic Acids Res. 37 (1), 1-13 (2009).</li><li>Ohta, S., 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=The+protein+composition+of+mitotic+chromosomes+determined+using+multiclassifier+combinatorial+proteomics.">The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics.</a> Cell. 142 (5), 810-821 (2010).</li><li>Yahi, 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=Differential+cooperation+between+heterochromatin+protein+HP1+isoforms+and+MyoD+in+myoblasts.">Differential cooperation between heterochromatin protein HP1 isoforms and MyoD in myoblasts.</a> J Biol Chem. 283 (35), 23692-23700 (2008).</li><li>Philipot, O., 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=The+core+binding+factor+CBF+negatively+regulates+skeletal+muscle+terminal+differentiation.">The core binding factor CBF negatively regulates skeletal muscle terminal differentiation.</a> PloS one. 5 (2), (2010).</li><li>Joliot, V., 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=The+SWI/SNF+subunit/tumor+suppressor+BAF47/INI1+is+essential+in+cell+cycle+arrest+upon+skeletal+muscle+terminal+differentiation.">The SWI/SNF subunit/tumor suppressor BAF47/INI1 is essential in cell cycle arrest upon skeletal muscle terminal differentiation.</a> PloS one. 9 (10), e108858 (2014).</li><li>Tanese, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+density+gradient+sedimentation+to+separate+and+analyze+multiprotein+complexes.">Small-scale density gradient sedimentation to separate and analyze multiprotein complexes.</a> Methods. 12 (3), 224-234 (1997).</li><li>Singh, K., 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+KAP1+phosphorylation+switch+controls+MyoD+function+during+skeletal+muscle+differentiation.">A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation.</a> Genes Dev. 29 (5), 513-525 (2015).</li><li>Cong, L., 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Yahi, H., Philipot, O., Guasconi, V., Fritsch, L., Ait-Si-Ali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modification+and+muscle+differentiation.">Chromatin modification and muscle differentiation.</a> Expert Opin Ther Targets. 10 (6), 923-934 (2006).</li><li>Sun, L., Liu, L., Yang, X. J., Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt+binds+prohibitin+2+and+relieves+its+repression+of+MyoD+and+muscle+differentiation.">Akt binds prohibitin 2 and relieves its repression of MyoD and muscle differentiation.</a> J Cell Sci. 117. 117 (Pt 14), 3021-3029 (2004).</li><li>Caretti, G., 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=The+RNA+helicases+p68/p72+and+the+noncoding+RNA+SRA+are+coregulators+of+MyoD+and+skeletal+muscle+differentiation.">The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation.</a> Dev Cell. 11 (4), 547-560 (2006).</li><li>Knoepfler, P. S., 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+conserved+motif+N-terminal+to+the+DNA-binding+domains+of+myogenic+bHLH+transcription+factors+mediates+cooperative+DNA+binding+with+pbx-Meis1/Prep1.">A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.</a> Nucleic Acids Res. 27 (18), 3752-3761 (1999).</li><li>Albini, S., Puri, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWI/SNF+complexes,+chromatin+remodeling+and+skeletal+myogenesis:+it's+time+to+exchange!.">SWI/SNF complexes, chromatin remodeling and skeletal myogenesis: it's time to exchange!.</a> Exp Cell Res. 316 (18), 3073-3080 (2010).</li><li>Yahi, H., Fritsch, L., Philipot, O., Guasconi, V., Souidi, M., Robin, P., Polesskaya, A., Losson, R., Harel-Bellan, A., Ait-Si-Ali, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Cooperation+between+Heterochromatin+Protein+HP1+Isoforms+and+MyoD+in+Myoblasts.">Differential Cooperation between Heterochromatin Protein HP1 Isoforms and MyoD in Myoblasts.</a> J Biol Chem. 283 (35), 23692-23700 (2008).</li></ol>

The authors have nothing to disclose.

O método apresentado permite a identificação exaustiva de parceiros de um fator de transcrição, MyoD. Revelou parceiros MyoD num sistema heterólogo resistente a diferenciação induzida por MyoD, nomeadamente células de HeLa-S3. Assim, por definição, MyoD parceiros identificados são expressos ubiquamente. Estes incluem factores gerais e específicos de sequência de transcrição, enzimas modificadoras da cromatina, proteínas de processamento de RNA, quinases (Tabelas 1 e 2). Dado que as células de HeLa-S3 são resistentes a trans-diferenciação induzida por MyoD, MyoD actividade é principalmente repressiva e os parceiros identificados são susceptíveis de ser MyoD co-repressores. Isto é realçado pela presença de proteínas Id (Tabelas 1 e 2, Figura 2), que inibem a MyoD capacidade transativação 8. É importante ressaltar que tal estado repressivo corresponde ao status MyoD em proliferando mioblastos. Na verdade, confirmou que as interações MyoD com CBFβ, um MyoDparceiro identificado pelo método descrito, podem ser detectados em mioblastos C2C12 proliferar, mas são perdidas quando as células foram submetidos a diferenciação (ver a Figura 3D). No entanto, é importante notar que uma das principais limitações da metodologia apresentada é a falta de identificação MyoD co-activadores. Consistentemente, usando este método de nenhum dos co-activadores MyoD conhecidos, tais como histona-acetiltransferases 32 foram identificados. Purificação de complexos MyoD, quer a partir da fracção solúvel (sal nuclear extraível, SE) do núcleo ou a fracção enriquecida em cromatina (nucleossoma enriquecido, NE) (Figura 1A), que serve, pelo menos, duas funções principais. Em primeiro lugar, tal fraccionamento aumenta a representação dos parceiros não estequiométricas que seriam mascarados se purificação MyoD realizada a partir de extractos nucleares totais. Em segundo lugar, isto permite a separação de duas subpopulações funcionais de MyoD: pré-depositados / EVICted (SE) e ligados a cromatina (NE). Entre interagentes específicas para MyoD DNA-desacoplado, muitas quinases, factores de transcrição, o tráfico de proteínas, bem como foram identificados alguns remodelers cromatina (Figura 2). Quando totalmente validado, uma rede deste tipo poderia fornecer uma visão sobre o modo de regulação de MyoD DNA-desacoplado atividade. Pesquisa adicional para MyoD modificações pós-traducionais nestes dois compartimentos nucleares por espectrometria de massa, poderia desvendar ainda mais regulação da atividade MyoD. Finalmente, o fraccionamento dos complexos solúveis e ligadas MyoD-cromatina em um gradiente de glicerol revelou que, enquanto a MyoD ligada a cromatina está contida principalmente em um complexo, MyoD ADN não ligado é distribuída em três sub-complexos (Figura 1B). Caracterização destes sub-complexos por qualquer MS e / ou Western blot contra os candidatos de proteína deve elucidar o modo de regulação MyoD. Como ressaltado acima, as células HeLa naturalmente não EXPREss o fator de transcrição músculo MyoD. Foi, portanto, importante para confirmar as interações encontradas em um modelo de músculo esquelético (Figura 3). Muitos relatórios confirmados em modelos relevantes tais interacções funcionais entre MyoD e alguns dos parceiros identificados no ensaio de TAP-tag apresentados. Este é, por exemplo, o caso de proibitina 33, DDX17 34, MEIS1 35, CBF 27, HP1 26 e SWI / SNF 36,28,30 complexo remodelação da cromatina. Note-se que é possível efectuar tal purificação TAP-tag da baixa quantidade de células, tais como a partir de mioblastos, quando combinados com MS sensíveis. De fato, um estudo recente descreveu parceiros MyoD caracterização após a expressão indutível de MyoD marcada com Flag em mioblastos 30. Desde estável e contínuo superexpressão MyoD em mioblastos é deletéria, uma abordagem alternativa seria adicionando as tags Bandeira-HA para o alelo (s) MyoD endógeno em mioblastosusando métodos de edição de genoma, tais como CRISPR-Cas9 31. Notavelmente, a adição da etiqueta (s) poderia potencialmente alterar a função da proteína e / ou associação com os parceiros de ligação, por conseguinte o local da etiqueta (N- ou C-terminal) deve ser escolhida com cuidado. Um ensaio funcional da proteína de fusão no sistema em causa deve ser realizado antes de purificação para TAP-tag para assegurar a proteína etiquetada é funcional. A imunoprecipitação de proteínas endógenas evita estes problemas, no entanto, apoia-se na disponibilidade de um anticorpo específico e de afinidade elevada, o que é raramente disponível. Outro valor acrescentado da abordagem descrita, é a possibilidade de identificação de modificações pós-traducionais (PTMs) da própria proteína purificada e dos seus parceiros abundantes. Assim, com este recurso a purificação TAP-tag é adequado para identificar não só novos parceiros de interação, mas também novas funções enzimáticas associadas à proteína de interesse e / ou seus parceiros. Dentrono caso de uma proteína de ligação da cromatina (ie., factores de transcrição, enzimas), este método é, assim, adaptado para a identificação do &quot;código de histona&quot; associado. Com efeito, as caudas das histonas amino-terminais, que estão expostas na superfície do nucleossoma, estão sujeitas a múltiplas PTMs covalentes. PTMs histonas conferem uma única assinatura para os nucleossomos envolvidos. Uma combinação de diferentes modificações nas caudas N-terminais das histonas pode assim alterar a estrutura da cromatina para permitir a regulação da expressão do gene. Assim, caracterizando tais modificações associadas a uma determinada proteína pode fornecer insights sobre as funções e mecanismos de ação das proteínas de ligação de cromatina estudados 22. Em resumo, a metodologia apresentada permite a identificação completa de parceiros MyoD. A purificação TAP-tag fornece uma alternativa a outras abordagens, tais como GST pull-baixos, ensaios de levedura de dois híbridos e de exibição em fagos. Mesmo que por razões práticas (production de grande quantidade de extractos nucleares), temos de usar um sistema celular heteróloga, temos sido capazes de confirmar a participação dos parceiros MyoD identificados na diferenciação do músculo esquelético. Os dados obtidos demonstraram que o factor miogénica MyoD parece interagir com uma variedade de proteínas que variam de reguladores da transcrição a proteínas de ligação de ARN, sugerindo que os diferentes mecanismos que regulam a actividade de um factor de transcrição. Em conclusão, a mesma metodologia pode ser utilizada para identificar parceiros ubiquamente expressas de numerosos factores nucleares que podem ser difíceis de estudar no seu contexto celular específica.

organismos multicelulares eucarióticos são compostas de diferentes órgãos e tecidos. Cada tecido funcional tem um padrão de expressão do gene específico, o qual é determinado em cada passo de diferenciação. Diferenciação celular envolve a activação de genes específicos, manutenção da sua expressão e, geralmente, o silenciamento de um conjunto de genes tais aqueles que estão envolvidos na proliferação celular. diferenciação de músculo esquelético, ou miogénese, é, assim, um processo multi-etapa, que começa com a determinação de células estaminais mesodermal em mioblastos, e, em seguida, leva à diferenciação terminal destes mioblastos em primeiro mono-nucleadas, e, em seguida, multi-nucleadas, miotubos . Assim, mioblastos são &quot;determinado&quot; células, que ainda são capazes de proliferar, mas eles estão comprometidos com a linhagem músculo esquelético, e, portanto, pode diferenciar exclusivamente em células musculares esqueléticas, quer durante o desenvolvimento embrionário ou em regeneração muscular adulta. O processo de terminal de músculo esquelético diferemciação é orquestrado por um programa genético específico que se inicia com a saída permanente do ciclo celular de células precursoras de mioblastos que leva a um silenciamento definitivo da proliferação de genes associados, tais como E2F genes alvo 1. Com efeito, durante o processo de diferenciação terminal, paragem proliferação de mioblastos é um passo crucial que precede a expressão de genes específicos do músculo esquelético e a fusão de mioblastos em miotubos 2. Tal programa permite que as células-tronco musculares adultas, também chamados de células satélites, para diferenciar durante o processo de regeneração após lesão do músculo esquelético. Myogenesis mamíferos é criticamente dependente de uma família de miogênica hélice-volta-hélice (bHLH) fatores de transcrição MyoD, Myf5, MRF4 e miogenina, frequentemente referida como a família de fatores esqueléticos determinação muscular ou MRFs (músculo fatores regulatórios) 3. Cada um deles desempenha um papel essencial na especificação e DIFdiferen- das células musculares esqueléticas e tem um padrão de expressão específico abril 05-07. A ativação de Myf5 e MyoD constitui o passo determinante que compromete as células da linhagem miogênica e subsequente expressão de miogenina desencadeia myogenesis com a ativação de genes específicos do músculo esquelético, como MCK (Muscle Creatina Quinase). Factores de transcrição bHLH Miogênica cooperar com os membros da família MEF2 na activação de genes musculares de loci anteriormente silenciosos 8. Eles também estimulam esquelético transcrição do gene muscular como heterodímeros com proteínas bHLH onipresente, E12 e E47, conhecidas como proteínas E, que se ligam chamados E-caixas em várias regiões do gene-regulação 8. Torção, Id (inibidor de diferenciação) e outros factores regulam negativamente este processo, por competir com MyoD para proteínas E de ligação 8. MyoD é considerado como o jogador mais importante no desencadeamento de terminal muscular diferenciação 9 </s-se&gt; uma vez que tem a capacidade de induzir um programa de determinação miogénica / diferenciação (trans-diferenciação), em muitos sistemas não-musculares completamente diferenciadas 10-13. Na verdade, a expressão forçada de MyoD induz a trans-diferenciação de diferentes tipos celulares, mesmo aqueles derivados de outra origem embriológica 12. Por exemplo, pode converter MyoD hepatócitos, f ibroblastos, melanócitos, neuroblastos e adipocitos em células musculares semelhante. A ação trans-de diferenciação de MyoD envolve uma ativação anormal do programa genético miogênica (nomeadamente os seus genes-alvo) em um ambiente não-muscular, concomitante ao silenciamento do programa genético de origem (nomeadamente, os genes de proliferação). Em proliferando mioblastos, MyoD é expresso, mas não é capaz de ativar seus genes-alvo, mesmo quando ele se liga aos seus promotores 14-16. Por conseguinte, o requisito de MyoD de ser continuamente expresso em mioblastos indiferenciadas permanece bastante Elusive. MyoD pôde reprimir seus genes-alvo devido ao recrutamento de enzimas modificadoras da cromatina repressivas em proliferando mioblastos antes do carregamento de ativar cromatina remodelação enzimas 14,17. Por exemplo, na proliferação mioblastos, MyoD está associada com o co-repressor de transcrição KAP-1, histona desacetilases (HDACs) e metiltransferases lisina repressivas (KMTS), incluindo a histona H3 lisina 9 ou H3K9 e H3K27 KMTS, e suprimir de forma activa a expressão de genes alvo através do estabelecimento de uma estrutura de cromatina 14,17 localmente repressivo. Importante, um relatório recente indicou que MyoD é em si directamente metilado pelo H3K9 KMT G9a resultando na inibição da sua actividade de transactivação 16. Os mecanismos epigenética envolvidas neste trans-diferenciação de células não musculares por MyoD são em grande parte desconhecida. Notavelmente, algumas linhas de células são resistentes a trans-diferenciação induzida por MyoD. Assim, em células HeLa, MyoD é ou inactiva ouainda pode funcionar como repressor, em vez de activador da transcrição devido à falta de expressão da subunidade BAF60C remodelação da cromatina complexo SWI / SNF 18. Este modelo pode, assim, ser de escolha para melhor caracterizar os mecanismos de repressão do gene induzida por MyoD. É igualmente apropriado para ensaiar a capacidade de MyoD para induzir ambiente cromatina repressivo na sua loci alvo com seus parceiros associados e, portanto, desvendar os mecanismos repressivos dependente de MyoD em proliferando mioblastos para afinar a diferenciação terminal. Aqui descrevemos o protocolo para a identificação de parceiros MyoD usando purificação de afinidade em tandem (TAP-tag) acoplada a espectrometria de massa (MS) caracterização. A utilização de células HeLa-S3 que expressam estavelmente Bandeira-HA-MyoD permitida para obter material suficiente para purificar os complexos a partir de extractos nucleares MyoD fraccionados. A identificação de parceiros MyoD no sistema heterólogo foi seguido por validatino em um sistema relevante.

<table border="0" cellpadding="0" cellspacing="0" width="543">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Cell lines</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HeLa-S3</td>
			<td style="width:115px;">ATCC</td>
			<td style="width:101px;">CCL-2.2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">C2C12</td>
			<td style="width:115px;">ATCC</td>
			<td style="width:101px;">CRL-1772</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Equipment</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Spinner</td>
			<td style="width:115px;">Corning</td>
			<td style="width:101px;">778531</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Homogenizer :&#160; Dounce homogenizer&#160;</td>
			<td style="width:115px;">Wheaton</td>
			<td style="width:101px;">432-1273 and 432-1271</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Agitating device for spinners</td>
			<td style="width:115px;">Bellco</td>
			<td style="width:101px;">778531</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Sonicator</td>
			<td style="width:115px;">Diagenode</td>
			<td style="width:101px;">UCD 200</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Hemocytometer</td>
			<td style="width:115px;">Marienfeld Superior&#160;</td>
			<td style="width:101px;">640610</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Low-binding tubes</td>
			<td style="width:115px;">Sorenson</td>
			<td style="width:101px;">27210</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Empty spin column</td>
			<td style="width:115px;">Bio-Rad</td>
			<td style="width:101px;">7326204</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Reagents</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">SDS-polyacrylamide 4-12 %&#160; gel</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0336BOX</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4X loading buffer&#160;</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0007</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">10X reducing agent</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0004</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Silver staining kit</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">A8592</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Centrifugal filter units, 10K</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:101px;">UFC501024</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protein G agarose beads</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">P4691</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Protein A/G&#160; Resin</td>
			<td style="width:115px;">Thermo Scientific</td>
			<td style="width:101px;">53132</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Flag resin (anti-Flag M2-agarose affinity gel)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2220</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">HA resin (monoclonal Anti-HA agarose)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A2095</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MNase</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">N3755</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Flag free peptide (DYKDDDDK)</td>
			<td style="width:115px;">Ansynth Service BV</td>
			<td style="width:101px;">Custom synthesis</td>
			<td>Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">HA free peptide (YPYDVPDYA)</td>
			<td style="width:115px;">Ansynth Service BV</td>
			<td style="width:101px;">Custom synthesis</td>
			<td>Resuspend up to 4 mg/mL in 50 mM Tris-HCl, pH 7.8</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Bicinchoninic acid based protein assay kit : BCA kit</td>
			<td style="width:115px;">Thermo Scientific</td>
			<td style="width:101px;">23225</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Protease inhibitors</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S8830</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:177px;">Luminol-based enhanced chemiluminescence (ECL) HRP substrate</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">34075</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BSA</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9647</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">Sheared salmon sperm DNA (Deoxyribonucleic acid sodium salt from salmon testes)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D1626</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Spermine tetrahydrochloride</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S1141</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Spermidine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">S0266</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">G5516</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBS</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D8537</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MOPS running buffer</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">NP0001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM (high glucose)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">D0822</td>
			<td>Pre-warm&#160; at 37 &#176;C before use</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Trypsin-EDTA (0.05% phenol red</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;">Serum</td>
			<td>GE healthcare life sciences&#160;</td>
			<td style="width:101px;">PAA A15-102</td>
			<td style="width:151px;">Each lot of serum has to be first tested for your cells.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and Streptomycin&#160;</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:115px;">Life technologies</td>
			<td style="width:101px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Water (sterile-filtered)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">W3500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Name</td>
			<td style="width:115px;">Company</td>
			<td style="width:101px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Antibodies</td>
			<td style="width:115px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HA from rat (12CA5)</td>
			<td style="width:115px;">Roche</td>
			<td style="width:101px;">11583816001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Flag</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A8592</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MyoD</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>sc-32758</td>
			<td>To use for western blotting</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">MyoD</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>SC-760</td>
			<td>To use for immunoprecipitation and western blotting</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">CBFbeta</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">FL-182&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1alpha</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">2HP2G9</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1beta</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">1MOD1A9AS</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">HP1gamma</td>
			<td style="width:115px;">Euromedex</td>
			<td style="width:101px;">2MOD1GC</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:177px;">Suv39h1</td>
			<td style="width:115px;">Cell Signaling Technology</td>
			<td style="width:101px;">8729</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">BAF47</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:101px;">612111</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Myf5</td>
			<td style="width:115px;">Santa Cruz</td>
			<td>SC-302</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Tubulin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">T9026</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Actin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A5441</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">IgG Mouse</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">SC-2025</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">IgG Rabbit</td>
			<td style="width:115px;">Santa Cruz</td>
			<td style="width:101px;">SC-2027</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-rat -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A9037</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-rabbit -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A6154&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:177px;">Goat anti-mouse -HRP</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:101px;">A4416</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of myod interactome using tandem affinity purification coupled to mass spectrometry

diferenciação terminal músculo esquelético começa com o compromisso de células precursoras pluripotentes mesodermais de mioblastos. Estas células ainda tem a capacidade de proliferar ou eles podem se diferenciar e se fundem em miotubos multinucleadas, que maturate ainda mais para formar fibras musculares. diferenciação terminal músculo esquelético é orquestrada pela ação coordenada de vários fatores de transcrição, em particular os membros dos fatores ou MRFs reguladoras muscular (MyoD, miogenina, Myf5 e MRF4), também chamado de bHLH miogênica fatores de transcrição da família. Estes factores cooperar com complexos de remodelação da cromatina dentro da rede reguladora da transcrição elaborada para atingir tecido muscular esquelético. Neste, MyoD é considerado o fator de transcrição miogênica mestre no desencadeamento de diferenciação terminal muscular. Esta noção é reforçada pela sua capacidade de converter MyoD células não musculares em células do músculo esquelético. Aqui nós descrevemos uma abordagem utilizada para identificar MyoDparceiros de proteína de forma exaustiva, a fim de elucidar os diferentes fatores envolvidos na diferenciação terminal músculo esquelético. O objectivo a longo prazo é compreender os mecanismos epigenéticos envolvidos na regulação de genes musculares esqueléticas, ie., Metas MyoD. parceiros MyoD são identificados usando purificação de afinidade em tandem (TAP-tag) a partir de um sistema heterólogo acoplada a espectrometria de massa de caracterização (MS), seguido de uma validação do papel de parceiros relevantes durante a diferenciação terminal de músculo esquelético. formas aberrantes de factores miogénicos, ou a sua regulação aberrante, está associada com um número de desordens musculares: miastenia congénita, distrofia miotónica, rabdomiossarcoma e defeitos na regeneração muscular. Como tal, os factores miogï¿½icas fornecer um conjunto de potenciais alvos terapêuticos em doenças musculares, tanto no que diz respeito aos mecanismos que causam doença em si e os mecanismos regenerativos que pode melhorar o tratamento da doença. Assim, o understan detalhadaDing das interacções intermoleculares e os programas genéticos controlados pelos factores miogï¿½icas é essencial para a concepção racional de terapias eficientes.

Para compreender a regulação da actividade MyoD, realizamos a caracterização exaustiva dos complexos MyoD usando purificação bioquímica, com base no imunopurificação de uma forma marcada com o dobro da MyoD seguido por espectrometria de massa (MS). A utilização da linha de células de HeLa-S3 que expressa Flag-MyoD marcada com HA e uma linha de células de controlo que expressam HA-autorizações de bandeira para obter material suficiente para purificar o complexo MyoD efectuando a purificação por afinidade de duplo-Flag-HA-MyoD. Nós fracionado os extratos de células em frações citoplasmáticos e nucleares, em seguida, mais fraccionado a fração nuclear para extraído-sal (sal-extraível nuclear, SE) e enriquecida para nucleossomos (, enriquecido com nucleossomos NE) frações. Purificação TAP-Tag a partir destas fracções nucleares separados (Figura 1, as Tabelas 1 e 2) permitiu desvendar parceiros que têm relativamente baixa abundance quando localizada num compartimento subnuclear específica. Além disso, tal estratégia foi explorada para descobrir parceiros de desacoplado (SE) versus (NE) MyoD ligado ao ADN para obter insights sobre a regulação da atividade MyoD. Para a purificação TAP-Tag, foi utilizado o sistema em tandem epitopo Flag-HA. A pequena bandeira hidrofílica e epitopos HA tem o mínimo de interferência com a função da proteína e são altamente acessível para interação antígeno-anticorpo. Anti-Flag e anti-HA imunopurificação sequencial à base de resina foi realizada, seguida de eluição dos complexos imunopurificados utilizando péptidos da bandeira e HA. As proteínas eluídas foram então executados sobre um gel de SDS-PAGE para permitir que todas as proteínas de entrar no gel. Os pedaços de gel contendo todas as proteínas purificadas foram cortados; as proteínas foram extraídas, e identificado por espectrometria de massa (MS) digerido com tripsina. Como mostrado na Figura 2, </strong&gt; complexos MyoD purificadas de fração NE têm maior enriquecimento em fatores de transcrição e co-repressores. A análise MS desvendado uma série de parceiros conhecidos de MyoD (tais como PBX, Id, E12 / E47 (HTF4), BRG1 (SMCA4), MEIS1 ...) e novos parceiros que foram confirmados nos estudos gerados por esta análise (como o HP1, CBF, MBD3, BAF47 (SNF5 / INI1) e todas as outras subunidades de complexos de SWI / SNF ...) (Figura 2 e 3) 26-28. Isto lança luz sobre parceiros MyoD ligados a DNA e possíveis co-reguladores que podem estabelecer um ambiente repressivo-cromatina. Por exemplo, as proteínas HP1, que foram identificados como parceiros MyoD por metodologia descrita, são conhecidos por se ligarem H3K9 metilado para manter a repressão do gene e estrutura heterocromatina. Com efeito, o HP1 inibe a actividade de transcrição MyoD resultando em expressão do gene alvo MyoD prejudicada e músculo diferenciação terminal 26. Fraccionamento adicional deos complexos MyoD SE e NE em gradiente de glicerol (como descrito em 29) descobriram os MyoD sub-complexos nos dois compartimentos subnucleares (Figura 1B). Em particular, SE MyoD é distribuída em três sub-complexos, enquanto o MyoD ligada a cromatina pertence principalmente a um complexo. Algumas das TAP-tag / MS revelou interagentes foram confirmados por western blot em complexos MyoD. Estes incluem a CBF fator de transcrição, EBB, MTG8R eo BAF47 SWI / SNF subunidade (SNF5) (Figura 3A, à esquerda) e proteínas HP1 (CBX1 e CBX3) (Figura 3A, à direita). Importante, uma vez que as células HeLa que não são células musculares e não expressam MyoD, é necessário confirmar interacções entre interactores recentemente identificados e MyoD em mioblastos BD (Figura 3). Notavelmente, por essa validação, os extractos nucleares totais (sem separação em SE e NE) são geralmente suficientes, which permite a redução da quantidade de mioblastos utilizados para a preparação da amostra. Os ensaios de interação in vitro, em 26,27, ajudam a validar esses achados. Finalmente, os significados funcionais destas interações em células musculares devem ser abordadas como em 26-28. Tomados em conjunto, apresentou dados mostram uma visão global de parceiros de MyoD ubiquamente expressas e pavimentar o caminho para novos estudos funcionais em um modelo muscular mais relevante. <img alt="figura 1" src="/files/ftp_upload/53924/53924fig1.jpg" /> Figura 1: MyoD complexos isolados por afinidade em tandem de purificação (a) Uma coloração com prata dos complexos eMyoD purificados por afinidade duplas isoladas a partir de sal-extraível nuclear (SE) ou nucleossoma enriquecido (NE) fracções nucleares de linhas de células HeLa-S3 estável. expressando Bandeira-HA-MyoD (complexo MyoD) umlinha celular de controlo d (Mock). MW, marcador de peso molecular em daltons quilo (kDa). A seta indica Bandeira-HA-MyoD (eMyoD). Esta pesquisa foi publicada originalmente em 37. Copyright A Sociedade Americana de Bioquímica e Biologia Molecular. Este valor foi modificado a partir de 26: as pistas com purificações simulados e eMyoD complexo isolado de frações nucleares SE são agora mostrados. (B) eMyoD complexos purificados por afinidade dupla como em (A) foram fraccionados em gradiente de glicerol varia desde 20% a 41%. As fracções foram recolhidas manualmente, concentrados e analisados ​​por Western blot (WB) utilizando anticorpos anti-FLAG. Observe a presença de vários eMyoD contendo sub-complexos na fração nuclear de sal-extraível (SE). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/53924fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 2" src = &quot;/ files / ftp_upload / 53924 / 53924fig2.jpg&quot; /&gt; Figura 2:. Comparação de cromatina-bound (nucleossomo enriquecido, NE) versus Solúvel Nuclear (sal nuclear extraível, SE) MyoD Parceiros Top: Venn diagrama mostrando sobreposição entre interactianos eMyoD isoladas de sal-extraível nuclear (SE) e enriqueceu-nucleossomo ( NE) fração. As proteínas ribossomais, factores de iniciação de tradução, factores de reparação de ADN e as isoformas de tubulina foram excluídos da análise de como eles estão presentes em diversos conjuntos diferentes de dados obtidos pela TAP e considerados não-específica inferior:. Os interactores MyoD encontrados tanto em SE e frações NE (comuns) ou específicas para uma das fracções (únicas) foram divididos em grupos com base em suas anotações funcionais. Citoesqueleto relacionadas com outras proteínas e variado não estão representados. As proteínas sublinhadas são interagentes MyoD validadas tanto em HeLa e / ou em myoblasts por co-imunoprecipitação. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/53924fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53924/53924fig3.jpg" /> Figura 3:. Validação do conjunto selecionado de MyoD interactianos (A) Validação de interagentes MyoD, identificado por espectrometria de massa, na linha de células HeLa-S3 expressando estavelmente Bandeira-HA-MyoD (eMyoD) Painel esquerdo:. Análise de Western blot de afinidade de casal complexos MyoD -purified isoladas de sal-extraível nuclear (SE) ou (NE) frações enriquecidas com nucleossomos de linha de células HeLa-S3 expressando estavelmente linha eMyoD ou célula de controlo (Mock) com os anticorpos indicados. MW, marcador de peso molecular Painel direito:. Análise de Western blot de complexos MyoD duplas purificados por afinidade isolado a partir de fracções enriquecidas com nucleossomos nucleares de HeLa-S3 linha de células svitavelmente expressar ou linha celular de controlo eMyoD (Mock) com anticorpos indicados. Este painel foi originalmente publicado no The Journal of Biological Chemistry. Yahi H, Fritsch L, Philipot O, Guasconi V, Souidi M, Robin P, Polesskaya A, Losson R, Harel-Bellan A, Ait-Si-Ali S. J Biol Chem. 2008 29 de agosto; 283 (35): 23692-700. doi: 10,1074 / jbc.M802647200. Epub 2008 Jul 2. Direitos Autorais A Sociedade Americana de Bioquímica e Biologia Molecular. Este valor foi modificado a partir de 26: tipo e tamanho de polícia foi alterada eo texto foi rodado para unificar a rotulagem no âmbito da figura. MyoD foi etiquetado como eMyoD para evitar a confusão com IP endógena apresentado nos outros painéis. (BD) Validação de interagentes MyoD em mioblastos de rato C2C12. (B) extratos totais nucleares de proliferação mioblastos C2C12 foram utilizadas para imunoprecipitação (IP) com anticorpos produzidos contra BAF47 (SNF5) ou MyoD, ou com IgG de controlo. Os precipitados resultantes foram analisados ​​por wit WBh os anticorpos indicados. Para anticorpos anti-MyoD mais longos (longa exposição.) E menor (expo curta.) Tempos de exposição são mostrados. extratos de entrada foram carregadas para mostrar os níveis de proteínas endógenas. Este painel foi publicado em 28 sob a licença Creative Commons Attribution (CC BY) (http://creativecommons.org/licenses/by/4.0/). Este valor foi modificado a partir de 28: tipo e tamanho polícia foi alterado e o teste foi rodado para unificar a rotulagem no âmbito da figura. (C) Os extractos totais nuclear a partir de mioblastos C2C12 em proliferação foram utilizadas para imunoprecipitação com anticorpos criados contra MyoD, Suv39h1 (controlo positivo), ou IgG de controlo (controlo negativo). Os precipitados resultantes foram então submetidas a WB com os anticorpos indicados. Este painel foi originalmente publicado no The Journal of Biological Chemistry. Yahi H, Fritsch L, Philipot O, Guasconi V, Souidi M, Robin P, Polesskaya A, Losson R, Harel-Bellan A, Ait-Si-Ali S. J Biol Chem. 2008 29 de agosto; 283(35): 23692-700. doi: 10,1074 / jbc.M802647200. Epub 2008 Jul 2. Direitos Autorais A Sociedade Americana de Bioquímica e Biologia Molecular. Este valor foi modificado a partir de 26: tipo e tamanho de polícia foi alterada eo texto foi rodado para unificar a rotulagem no âmbito da figura. (D) Os extractos totais nucleares de proliferar (prolif.) E diferenciadoras C2C12 de mioblastos (48 h, indicada como Diff.) Foram utilizados para imunoprecipitação (IP) com os anticorpos criados contra MyoD e Myf5, ou com IgG de coelho normal e com pérolas vazias quanto controle negativo. Os precipitados resultantes foram analisados ​​por WB com os anticorpos indicados. extratos de entrada foram carregadas para mostrar os níveis de proteínas endógenas. Este painel foi publicado em 27 sob a licença Creative Commons Attribution (CC BY) (http://creativecommons.org/licenses/by/4.0/). Este valor foi modificado a partir de 27: tipo e tamanho de polícia foi alterada eo texto foi rodado para unificar a rotulagem no âmbito da figura. Os painéis com os resultados obtidos com a proliferar e diferenciar C2C12 são separados em dois, em oposição à figura original. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/53924fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Tabela 1:. Lista de proteínas identificadas por análise de MS em Complexos eMyoD Duplo Affinity purificadas isoladas a partir Nuclear Fraction Salt-extraível após a subtração das proteínas de fundo (proteínas identificadas pelo MS em eluatos de linha celular de controlo foram consideradas como pano de fundo não específica .) os dados representam a soma de quatro purificações independentes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/Table_1.xlsx" target="_blank">por favor clique aqui para baixar esse arquivo.</a> Tabela 2: Lista de Proteínas Identified por Análise MS em Complexos eMyoD Duplo Affinity purificadas isoladas a partir Fraction Nucleosome enriquecido após a subtração das proteínas de fundo. Os dados representam a soma de três purificações independentes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/Table_2.xlsx" target="_blank">Por favor clique aqui para baixar esse arquivo.</a> Tabela 3. O tampão de composições. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/Table_3.xls" target="_blank">Por favor clique aqui para baixar esse arquivo.</a>

Watch this Scientific Journal Video about Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry at JoVE.com

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Identificação de MyoD interactome Usando purificação de afinidade em tandem Acoplado a Espectrometria de Massa

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

identification-myod-interactome-using-tandem-affinity-purification

Nanyang Technological University

Research

JoVE Journal

Biology

9.8K visualizações.  Centre National de la Recherche Scientifique CNRS - Université Paris Diderot, Sorbonne Paris Cité. MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Vídeo: Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI preparação da amostra: o método de Camada Ultra Thin

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biologia

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

A avaliação quantitativa do Lipidome fermento usando Electrospray Espectrometria de Massa de Ionização

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantificação de Proteínas Usando Enriquecimento imunoafinidade Peptide Acoplado com Espectrometria de Massa

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Purificação de Proteínas ortogonais Facilitado por um Tag Affinity Pequenas Bispecific

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificação de parceiros de proteína Interagir utilizando Tandem purificação por afinidade

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Proteome Redox tiol Usando Isótopos Espectrometria de Massa Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Um protocolo para a identificação de interações proteína-proteína Baseado<sup&gt; 15</sup&gt; Etiquetagem N Metabólica, imunoprecipitação, Quantitativa Espectrometria de Massa e Affinity Modulação

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimento de purificação de afinidade com tag Proteínas Recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...