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>
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The authors have nothing to disclose.

Il metodo presentato consenta l&#39;identificazione esaustivo di partner di un fattore di trascrizione, MyoD. Ha rivelato partner MyoD in un sistema eterologo resistente alla differenziazione MyoD indotta, ovvero cellule HeLa-S3. Così, per definizione, i partner MyoD identificati sono ubiquitariamente espressi. Questi includono fattori generali e specifici di sequenza di trascrizione, enzimi cromatina modificanti, proteine ​​di elaborazione RNA, chinasi (Tabelle 1 e 2). Dato che le cellule HeLa-S3 sono resistenti a MyoD indotta trans-differenziazione, l&#39;attività MyoD è prevalentemente repressivo e le parti individuate sono suscettibili di essere MyoD co-repressori. Ciò è evidenziato dalla presenza di proteine ​​Id (Tabelle 1 e 2, Figura 2), noti per inibire MyoD capacità transattivazione 8. È importante sottolineare che tale stato repressivo corrisponde allo stato MyoD in proliferanti mioblasti. In effetti, abbiamo confermato che le interazioni con MyoD CBFβ, un MyoDPartner identificato con il metodo descritto, può essere rilevato in proliferanti mioblasti C2C12, ma vengono persi quando le cellule sono stati sottoposti differenziazione (vedi Figura 3D). Tuttavia, è importante notare che uno dei principali limiti del metodo presentato è la mancanza di MyoD identificazione co-attivatori. Coerentemente, utilizzando questo metodo nessuna delle note co-attivatori MyoD, come istone acetiltransferasi 32 sono stati identificati. Purificazione di complessi MyoD sia dalla frazione solubile (sale nucleare estraibile, SE) del nucleo o dalla frazione cromatina arricchita (nucleosoma arricchito, NE) (Figura 1A) serve per almeno due funzioni principali. In primo luogo, tale frazionamento aumenta la rappresentanza delle parti non stechiometriche che verrebbero mascherati se la purificazione MyoD eseguita da estratti nucleari totali. In secondo luogo, questo permette la separazione delle due sottopopolazioni funzionali di MyoD: pre-depositato / EVICTed (SE) e della cromatina-bound (NE). Tra interattori specifici per MyoD DNA non legato, molti chinasi, fattori di trascrizione, il traffico di proteine ​​così come sono stati individuati alcuni rimodellatori della cromatina (Figura 2). Quando è completamente convalidato, una tale rete potrebbe fornire uno spaccato del modo di regolazione dell&#39;attività di MyoD DNA non legato. Ricerca aggiuntiva per MyoD modificazioni post-traslazionali in questi due compartimenti nucleari mediante spettrometria di massa, potrebbe svelare un&#39;ulteriore regolamentazione dell&#39;attività MyoD. Infine, frazionamento di complessi MyoD solubili e cromatina-bound su un gradiente di glicerolo ha rivelato che mentre la MyoD cromatina-bound è principalmente contenuta in un unico complesso, MyoD DNA legato viene distribuito in tre sotto-complessi (Figura 1B). La caratterizzazione di questi sotto-complessi di entrambi MS e / o Western blot contro i candidati di proteine ​​dovrebbe chiarire ulteriormente il modo di regolazione MyoD. Come sottolineato in precedenza, le cellule HeLa non è naturalmente Express il fattore di trascrizione muscolo MyoD. Era pertanto importante confermare le interazioni trovati in un modello muscolo scheletrico (Figura 3). Molti rapporti confermati nei modelli rilevanti tali interazioni funzionali tra MyoD e alcuni dei partner individuati nel test TAP-tag presentato. Questo è per esempio il caso di proibitina 33, DDX17 34, Meis1 35, CBF 27, HP1 26, e SWI / SNF cromatina-rimodellamento 36,28,30 complesso. Si noti che è possibile eseguire tale purificazione TAP-tag dalla bassa quantità di cellule, ad esempio da mioblasti, se combinati a MS sensibili. Infatti, un recente documento descritto partner MyoD caratterizzazione dopo l&#39;espressione inducibile di MyoD Flag-tag nei mioblasti 30. Dato stabile e continuo sovraespressione MyoD nei mioblasti è deleteria, un approccio alternativo sarebbe aggiungendo i tag Flag-HA nel allele endogena MyoD (s) nei mioblastiutilizzando metodi genoma-editing, come CRISPR-Cas9 31. In particolare, l&#39;aggiunta del tag (s) potrebbe potenzialmente alterare la funzione delle proteine ​​e / o associazione con partner di legame, quindi il luogo del tag (N- o C-terminale) deve essere scelto con cautela. Un test funzionale della proteina di fusione nel sistema in questione deve essere eseguito prima di purificazione per TAP-tag per garantire la proteina tag è funzionale. Immunoprecipitazione di proteine ​​endogene evita questi problemi, tuttavia, si basa sulla disponibilità di un anticorpo specifico ed alta affinità, che è raramente disponibile. Un altro valore aggiunto dell&#39;approccio descritto è la possibilità di identificare modificazioni post-traduzionali (PTM) della proteina purificata stesso e dei suoi partner abbondanti. In tal modo, con questa caratteristica la purificazione TAP-tag è adatto per identificare non solo i nuovi partner di interazione, ma anche nuove funzioni enzimatiche associate alla proteina di interesse e / o ai suoi partner. Incaso di cromatina-binding protein (es., fattori di trascrizione, enzimi), questo metodo è pertanto adatta per l&#39;identificazione del &quot;codice istone&quot; associato. In effetti, le code degli istoni amino-terminale, che sono esposte sulla superficie nucleosoma, sono soggette a molteplici PTM covalenti. PTM istone conferiscono una firma unica per i nucleosomi coinvolti. Una combinazione di diverse modifiche sul istoni N-terminale code può quindi alterare la struttura della cromatina per permettere regolazione dell&#39;espressione genica. Così, che caratterizzano tali modifiche associati ad una data proteina potrebbe fornire approfondimenti sui ruoli e meccanismi di azione delle proteine ​​della cromatina vincolante studiati 22. In sintesi, la metodologia presentata consenta l&#39;identificazione completa del partner MyoD. La purificazione TAP-tag fornisce un&#39;alternativa ad altri approcci, come GST pull-down, lievito saggi a due ibridi e di visualizzazione dei fagi. Anche se per motivi pratici (production di grandi quantità di estratti nucleari) dobbiamo usare un sistema cellulare eterologo, siamo stati in grado di confermare la partecipazione delle parti MyoD identificati nella differenziazione muscolo scheletrico. I dati ottenuti dimostrano che il fattore miogenico MyoD sembra interagire con una pletora di proteine ​​che vanno da regolatori trascrizionali alle proteine ​​leganti RNA, suggerendo i diversi meccanismi che regolano l&#39;attività di un fattore di trascrizione. In conclusione, lo stesso approccio metodologico potrebbe essere utilizzato per individuare partner ubiquitariamente espressi di numerosi fattori nucleari che potrebbero essere difficili da studiare nel loro specifico contesto cellulare.

organismi pluricellulari eucarioti sono composti da diversi organi e tessuti. Ogni tessuto funzionale ha un pattern di espressione genica specifica, che è determinato ad ogni passo differenziazione. differenziazione cellulare coinvolge l&#39;attivazione di geni specifici, manutenzione della loro espressione e, in generale, il silenziamento di una serie di geni come quelli coinvolti nella proliferazione cellulare. Scheletrico differenziamento muscolare, o miogenesi, è quindi un processo a più fasi, che inizia con la determinazione delle cellule staminali mesodermiche in mioblasti, e poi porta alla differenziazione terminale di questi mioblasti in primo mono-nucleate, e quindi multi-nucleate, miotubi . Così, i mioblasti vengono &quot;determinate&quot; Le cellule, che sono ancora in grado di proliferare, ma si sono impegnati a lignaggio muscolo scheletrico, e quindi in grado di differenziarsi solo in cellule del muscolo scheletrico sia durante lo sviluppo embrionale o nella rigenerazione del muscolo adulto. Il processo di terminale muscolo scheletrico differireziazione è orchestrato da uno specifico programma genetico che inizia con l&#39;uscita permanente dal ciclo cellulare delle cellule precursori mioblasti che conduce ad un silenziamento definitiva di proliferazione associati geni, come E2F geni target 1. Infatti, durante il processo di differenziazione terminale, arresto mioblasti proliferazione è un passo cruciale che precede l&#39;espressione di geni specifici muscolo scheletrico e la fusione dei mioblasti in miotubi 2. Tale programma permette cellule staminali muscolari adulte, dette anche cellule satelliti, per differenziare durante il processo di rigenerazione dopo un trauma del muscolo scheletrico. Miogenesi mammiferi è criticamente dipendente da una famiglia di miogenico elica-ansa-elica (bHLH) fattori di trascrizione MyoD, Myf5, MRF4 e Myogenin, spesso indicato come la famiglia di fattori scheletrico determinazione muscolare o MRF (muscolo fattori normativi) 3. Ciascuno di essi ha un ruolo essenziale nella specifica e differenziazione delle cellule del muscolo scheletrico e ha uno specifico modello di espressione 4 5-7. L&#39;attivazione di Myf5 e MyoD costituisce il passo determinante che impegna le cellule al lignaggio miogenico, e la successiva espressione di Myogenin innesca miogenesi con l&#39;attivazione di geni specifici del muscolo scheletrico, come MCK (muscolo creatina chinasi). Myogenic fattori di trascrizione bHLH collaborano con i membri della famiglia MEF2 nel attivazione di geni muscolari da loci precedentemente silenziosa 8. Essi hanno inoltre stimolare scheletrico trascrizione genica muscolare eterodimeri con le proteine ​​onnipresente bHLH, E12 e E47, note come proteine ​​E, che legano i cosiddetti E-box in varie regioni del gene-normativo 8. Twist, Id (inibitore di differenziazione) e di altri fattori regolano negativamente questo processo, competendo con MyoD per le proteine ​​di legame E 8. MyoD è considerato come il giocatore importante nello scatenare muscolare terminale differenziazione 9 </sup&gt; poiché ha la capacità di indurre un programma determinazione miogenico / differenziazione (trans-differenziazione) in molti sistemi non-muscolari completamente differenziate 10-13. In effetti, l&#39;espressione forzata di MyoD induce la trans-differenziazione dei diversi tipi cellulari, anche quelli derivati ​​da un&#39;altra origine embryologic 12. Ad esempio, MyoD in grado di convertire gli epatociti, fibroblasti, melanociti, neuroblasti, e adipociti in cellule muscolari-like. L&#39;azione trans-differenziativo di MyoD comporta un&#39;attivazione anomala del programma genetico miogenico (in particolare i suoi geni bersaglio) in un ambiente non-muscolare, concomitante al silenziamento del programma genetico originale (in particolare, i geni della proliferazione). In proliferanti mioblasti, MyoD è espressa, ma non è in grado di attivare i suoi geni bersaglio anche quando si lega ai loro promotori 14-16. Pertanto, il requisito di MyoD siano continuamente espressi in mioblasti indifferenziati rimane abbastanza elusive. MyoD potrebbe reprimere i suoi geni bersaglio dovuto all&#39;assunzione di enzimi cromatina modificanti repressive in proliferanti mioblasti prima del carico di attivare il rimodellamento della cromatina enzimi 14,17. Per esempio, in proliferanti mioblasti, MyoD è associata a trascrizionale co-repressori KAP-1, istone deacetilasi (HDAC) e metiltransferasi lisina repressive (km in linea), tra cui l&#39;istone H3 lisina 9 o H3K9 e H3K27 km in linea, e sopprimere attivamente la sua espressione di geni bersaglio attraverso la definizione di un locale repressiva struttura della cromatina 14,17. È importante sottolineare che un recente rapporto ha indicato che MyoD è essa stessa direttamente metilato dalle H3K9 il KMT G9a con conseguente inibizione della sua attività transattivante 16. I meccanismi epigenetici coinvolti in questa trans-differenziazione delle cellule non muscolari di MyoD sono largamente sconosciuti. In particolare, alcune linee cellulari sono resistenti a MyoD indotta trans-differenziazione. Così, in cellule HeLa, MyoD è inattivo oaddirittura potrebbe funzionare come repressore piuttosto che attivatore della trascrizione a causa della mancanza di espressione della subunità BAF60C del rimodellamento della cromatina complesso SWI / SNF 18. Questo modello può quindi essere di scelta per caratterizzare meglio i meccanismi di MyoD indotta repressione gene. E &#39;adatto anche per saggiare la capacità di MyoD di indurre ambiente cromatina repressiva alla sua loci bersaglio con i suoi partner associati e quindi scoprire i meccanismi repressivi MyoD-dipendente della proliferazione mioblasti per mettere a punto la differenziazione terminale. Qui si descrive il protocollo per l&#39;identificazione dei partner MyoD utilizzando Tandem Affinity Purification (TAP-Tag) accoppiata alla spettrometria di massa caratterizzazione (MS). L&#39;uso di cellule HeLa-S3 esprimono stabilmente Flag-HA-MyoD consentito di ottenere abbastanza materiale per purificare i complessi MyoD da estratti nucleari frazionati. L&#39;identificazione di partner MyoD nel sistema eterologo è seguito validation in un sistema di riferimento.

<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

Scheletrico differenziazione terminale muscolare inizia con l&#39;impegno di cellule precursori mesodermiche pluripotenti a mioblasti. Queste cellule hanno ancora la capacità di proliferare o possono differenziarsi e si fondono in miotubi multinucleati, che maturare ulteriormente per formare fibre muscolari. Scheletrico differenziazione terminale muscolare è orchestrata con l&#39;azione coordinata di diversi fattori di trascrizione, in particolare i membri dei fattori regolatori muscolari o MRF (MyoD, Myogenin, Myf5, e MRF4), chiamato anche il miogena bHLH fattori di trascrizione della famiglia. Questi fattori cooperano con i complessi della cromatina-rimodellamento all&#39;interno della rete regolamentare trascrizionale elaborato per raggiungere miogenesi scheletrico. In questo, MyoD è considerato il maestro miogenico fattore di trascrizione nello scatenare differenziamento terminale muscolare. Questa nozione è rafforzata dalla capacità di MyoD convertire cellule non muscolari in cellule muscolari scheletriche. Qui si descrive un metodo utilizzato per identificare MyoDpartner proteici in modo esaustivo, al fine di chiarire i diversi fattori coinvolti nella scheletrico differenziazione terminale muscolare. L&#39;obiettivo a lungo termine è quello di comprendere i meccanismi epigenetici coinvolti nella regolazione dei geni muscolo scheletrico, vale a dire., Obiettivi MyoD. partner MyoD sono identificati utilizzando Tandem Affinity Purification (TAP-Tag) da un sistema eterologo accoppiato alla caratterizzazione spettrometria di massa (MS), seguito dalla validazione del ruolo delle parti interessate durante scheletrico differenziamento terminale muscolare. forme aberranti di fattori miogenici, o la loro regolamentazione aberranti, sono associati con una serie di disturbi muscolari: miastenia congenita, distrofia miotonica, rabdomiosarcoma e difetti nella rigenerazione muscolare. Come tale, i fattori miogenici forniscono un pool di potenziali bersagli terapeutici nei disturbi muscolari, sia per quanto riguarda i meccanismi che causano la malattia stessa e meccanismi rigenerativi che possono migliorare il trattamento della malattia. Così, il understan dettagliatading delle interazioni intermolecolari ei programmi genetici controllate dai fattori miogenici è essenziale per la progettazione razionale di terapie efficaci.

Per comprendere la regolazione dell&#39;attività MyoD, abbiamo intrapreso la caratterizzazione esaustivo dei complessi MyoD utilizzando purificazione biochimica, basato sulla immunopurificazione di una forma doppia targhetta di MyoD seguita da spettrometria di massa (MS). L&#39;uso della linea di cellule HeLa-S3 esprime MyoD Flag-HA-tag e una linea cellulare di controllo che esprime permessi Flag-HA per ottenere abbastanza materiale per purificare il complesso MyoD eseguendo doppio affinità purificazione di Flag-HA-MyoD. Abbiamo frazionato gli estratti di cellule in frazioni citoplasmatiche e nucleari, poi ulteriormente frazionato la frazione nucleare al sale estratti (sale-estraibili nucleari, SE) e arricchito per nucleosomi (NE, nucleosoma arricchito) le frazioni. TAP-Tag purificazione da queste frazioni nucleari separate (Figura 1, tabelle 1 e 2) è permesso svelare i partner che hanno relativamente basso abundance quando localizzato in uno specifico comparto subnucleare. Inoltre, tale strategia è stata sfruttata per scoprire partner non legato (SE) contro (NE) MyoD DNA-bound per ottenere approfondimenti sulla regolazione dell&#39;attività MyoD. Per la depurazione TAP-Tag, il sistema tandem epitopo Flag-HA è stato utilizzato. La piccola bandiera idrofila e epitopi HA hanno la minima interferenza con la funzione delle proteine ​​e sono altamente accessibili per l&#39;interazione antigene-anticorpo. Anti-Flag e anti-HA a base di resine immunopurificazione sequenziale è stata effettuata, seguita da eluizione dei complessi immunopurified con bandiera e HA peptidi. Le proteine ​​eluite sono state poi eseguite su una SDS-PAGE per consentire tutte le proteine ​​di entrare nel gel. I pezzi di gel contenenti tutte le proteine ​​purificate sono stati tagliati; le proteine ​​sono state estratte, tripsina digerito e identificati mediante spettrometria di massa (MS). Come mostrato in figura 2, </strong&gt; complessi MyoD purificati dalla frazione NE hanno una maggiore arricchimento di fattori di trascrizione e co-repressori. analisi MS svelato una serie di partner noti di MyoD (come Pbx, Id, E12 / E47 (HTF4), BRG1 (SMCA4), MEIS1 ...) e nuovi partner che sono stati confermati negli studi generati da questa analisi (come HP1, CBF, MBD3, BAF47 (SNF5 / INI1) e tutti gli altri subunità complesso SWI / SNF ...) (Figura 2 e 3) 26-28. Questo mette in luce i partner MyoD DNA-bound e possibili co-regolatori che possono stabilire un ambiente repressivo-cromatina. Ad esempio, le proteine ​​HP1, che sono stati identificati come partner MyoD di metodologia descritta, sono noti per legarsi H3K9 metilato per mantenere la repressione del gene e la struttura heterochromatin. Infatti, HP1 inibisce l&#39;attività trascrizionale MyoD con conseguente compromissione della espressione del gene bersaglio MyoD e terminale muscolare differenziazione 26. Ulteriore frazionamento dii complessi MyoD SE e NE su gradiente di glicerolo (come descritto in 29) scoperto sottosistemi complessi MyoD nei due compartimenti subnucleari (Figura 1B). In particolare, SE MyoD è distribuito in tre sub-complessi, mentre il MyoD cromatina-bound appartiene principalmente ad un unico complesso. Alcuni dei TAP-tag / MS ha rivelato interattori sono stati confermati da Western Blot su complessi MyoD. Questi includono il fattore di trascrizione CBF, EBB, MTG8R e la subunità BAF47 SWI / SNF (SNF5) (Figura 3A, a sinistra) e le proteine ​​HP1 (CBX1 e CBX3) (Figura 3a, a destra). È importante sottolineare che, poiché le cellule HeLa non sono cellule muscolari e non esprimono MyoD, è necessario confermare le interazioni tra interattori recentemente identificati e MyoD nei mioblasti (Figura 3 BD). In particolare, per tale convalida, gli estratti nucleari totali (senza separazione di SE e NE) sono in genere sufficienti, which consente la riduzione della quantità di mioblasti utilizzati per la preparazione del campione. I test in vitro di interazione come in 26,27, aiutano a validato ulteriormente questi risultati. Infine, i significati funzionali di queste interazioni in cellule muscolari dovrebbero essere ulteriormente trattate come in 26-28. Nel loro insieme, hanno presentato i dati mostrano una visione globale di partner MyoD ubiquitariamente espressi e aprire la strada ad ulteriori studi funzionali in un modello muscolo più rilevante. <img alt="Figura 1" src="/files/ftp_upload/53924/53924fig1.jpg" /> Figura 1: MyoD Complessi isolati da Tandem Affinity Purification (A) Una colorazione argento del doppio complessi eMyoD purificati per affinità isolati dal sale-estraibile nucleare (SE) o nucleosomi arricchito (NE) frazioni nucleari di linee di cellule HeLa-S3 stabile. esprimendo Flag-HA-MyoD (complesso MyoD) und linea cellulare di controllo (Mock). MW, marcatore di peso molecolare chilo Dalton (kDa). La freccia indica Flag-HA-MyoD (eMyoD). Questa ricerca è stato originariamente pubblicato in 37. Copyright L&#39;American Society for Biochimica e Biologia Molecolare. Questa cifra è stata modificata da 26: le corsie con purificazioni finte e eMyoD complesso isolato da frazioni nucleari SE sono ora mostrati. (B) doppia purificati per affinità complessi eMyoD come in (A) sono stati frazionati sul gradiente di glicerolo che vanno dal 20% al 41%. Le frazioni sono state raccolte manualmente, concentrati e analizzati mediante Western blot (WB) con anticorpi anti-Flag. Si noti la presenza di diversi eMyoD contenenti sotto-complessi in (SE) frazione nucleare sale-estraibili. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/53924fig1large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src = &quot;/ files / ftp_upload / 53924 / 53924fig2.jpg&quot; /&gt; Figura 2:. Confronto di cromatina-bound (nucleosomi arricchito, NE) rispetto solubile nucleare (sale nucleare estraibile, SE) MyoD Partners Top: diagramma di Venn che mostra sovrapposizione tra interattori eMyoD isolati dal sale-estraibile nucleare (SE) e nucleosoma arricchito ( NE) frazione. Le proteine ​​ribosomali, fattori di traduzione-iniziazione, fattori di riparazione del DNA e delle isoforme di tubulina sono stati esclusi dall&#39;analisi in quanto sono presenti in vari insiemi di dati diversi ottenuti da TAP e considerati come non specifico basso:. Gli interattori MyoD si trovano in entrambi SE e frazioni NE (comuni) o specifici per una delle frazioni (uniche) sono stati divisi in gruppi in base alle loro annotazioni funzionali. Citoscheletro-correlata e altre proteine ​​vari non sono raffigurati. Le proteine ​​sono sottolineate interattori MyoD convalidati sia in HeLa e / o in myoblasts di co-immunoprecipitazione. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/53924fig2large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53924/53924fig3.jpg" /> Figura 3:. Validazione di Selected Set di MyoD Interactiani (A) Convalida di interattori MyoD, identificato mediante spettrometria di massa, in linea di cellule HeLa-S3 esprimono stabilmente Flag-HA-MyoD (eMyoD) Pannello sinistro:. Analisi Western Blot di doppia affinità complessi MyoD -purified isolati dal sale-estraibile nucleare (SE) o (NE) frazioni nucleosoma-arricchito della linea cellulare HeLa-S3 esprimono stabilmente eMyoD o cellule di controllo di linea (Mock) con gli anticorpi indicati. MW, marcatore peso molecolare pannello di destra:. Analisi Western Blot di doppi complessi MyoD purificati per affinità isolato da frazioni nucleosoma-arricchito nucleari di HeLa-S3 linea cellulare sesprimendo tably o linea cellulare di controllo eMyoD (Mock) con anticorpi indicati. Questo pannello è stato originariamente pubblicato su 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. 29 agosto 2008; 283 (35): 23.692-700. doi: 10,1074 / jbc.M802647200. Epub 2008 luglio 2. Copyright L&#39;American Society for Biochimica e Biologia Molecolare. Questa cifra è stata modificata da 26: il tipo e la dimensione della polizia è stato modificato e il testo è stato ruotato di unificare l&#39;etichettatura all&#39;interno della figura. MyoD è stato etichettato come eMyoD per evitare la confusione con IP endogena presentati negli altri pannelli. (BD) Validazione di interattori MyoD in C2C12 mioblasti di topo. (B) estratti totali nucleari proliferazione mioblasti C2C12 sono stati utilizzati per immunoprecipitazione (IP) con anticorpi contro BAF47 (SNF5) o MyoD, o con IgG di controllo. I precipitati risultanti sono stati analizzati con spirito WBh gli anticorpi indicati. Per gli anticorpi anti-MyoD più lunghi (lungo expo.) E più corta (short expo.) Sono riportati i tempi di esposizione. estratti di ingresso sono stati caricati per mostrare i livelli di proteina endogena. Questo pannello è stato pubblicato in 28 sotto la Creative Commons Attribution (CC BY) licenza (http://creativecommons.org/licenses/by/4.0/). Questa cifra è stata modificata da 28: tipo e dimensione di polizia è stata cambiata e il test è stato ruotato di unificare l&#39;etichettatura all&#39;interno della figura. (C) estratti totali nucleari proliferazione mioblasti C2C12 sono stati utilizzati per immunoprecipitazione con anticorpi contro MyoD, Suv39h1 (controllo positivo), o controllare IgG (controllo negativo). I precipitati ottenuti sono stati poi sottoposti a WB con gli anticorpi indicati. Questo pannello è stato originariamente pubblicato su 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. 29 agosto 2008; 283(35): 23.692-700. doi: 10,1074 / jbc.M802647200. Epub 2008 luglio 2. Copyright L&#39;American Society for Biochimica e Biologia Molecolare. Questa cifra è stata modificata da 26: il tipo e la dimensione della polizia è stato modificato e il testo è stato ruotato di unificare l&#39;etichettatura all&#39;interno della figura. (D) estratti totali nucleari proliferazione (prolif.) E differenziando C2C12 mioblasti (48 ore, indicato come Diff.) Sono stati utilizzati per immunoprecipitazione (IP) con anticorpi contro MyoD e Myf5, o con normale IgG di coniglio e con perline vuote come controllo negativo. I precipitati ottenuti sono stati analizzati mediante WB con gli anticorpi indicati. estratti di ingresso sono stati caricati per mostrare i livelli di proteina endogena. Questo pannello è stato pubblicato nel 27 sotto la Creative Commons Attribution (CC BY) licenza (http://creativecommons.org/licenses/by/4.0/). Questa cifra è stata modificata da 27: il tipo e la dimensione della polizia è stato modificato e il testo è stato ruotato di unificare l&#39;etichettatura all&#39;interno del fIGURA. I pannelli con i risultati ottenuti dal proliferare e differenziare C2C12 sono separati in due in contrapposizione alla figura originale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/53924fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Tabella 1:. Elenco delle proteine ​​identificate da MS Analysis in complessi eMyoD doppio purificate per affinità Isolato da nucleare Frazione Salt-estraibile dopo la sottrazione delle proteine ​​sfondo (proteine ​​identificate da MS in eluati da linea di cellule di controllo sono stati considerati come sfondo non specifico .) I dati rappresentano la somma di quattro purificazioni indipendenti. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/Table_1.xlsx" target="_blank">cliccate qui per scaricare questo file.</a> Tabella 2: Elenco delle proteine ​​Identified da MS Analysis in complessi eMyoD doppio purificate per affinità isolate dalla frazione Nucleosome arricchito dopo la sottrazione delle proteine ​​di sfondo. I dati rappresentano la somma delle tre purificazioni indipendenti. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/Table_2.xlsx" target="_blank">Cliccate qui per scaricare questo file.</a> Tabella 3. Buffer composizioni. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53924/Table_3.xls" target="_blank">Cliccate qui per scaricare questo file.</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.

Identificazione di MyoD Interattoma Utilizzando Tandem Affinity Purification accoppiato alla spettrometria di 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 Views.  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.

Video: 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 Preparazione del campione: il metodo ultra sottile strato

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

Ricerca

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.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

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.

Una valutazione quantitativa del Lipidome lievito con ionizzazione Electrospray Spettrometria di massa

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.

Quantificazione delle proteine ​​Utilizzando Arricchimento Peptide immunoaffinità Accoppiato con spettrometria di 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.

Purificazione delle proteine ​​ortogonali Facilitato da una piastrina Affinity 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.

Individuazione di partner proteine ​​interagenti mediante 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 ...

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 tiolo Proteome Redox Utilizzando tag spettrometria di massa isotopica

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.

Identificazione di complessi proteici in<em&gt; Escherichia coli</em&gt; Con sequenziale purificazione per affinità Peptide in combinazione con Tandem Mass Spectrometry

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.

Un protocollo per l&#39;identificazione di interazioni proteina-proteina Sulla base di<sup&gt; 15</sup&gt; N marcatura metabolica, immunoprecipitazione, Spettrometria di massa quantitativa e di affinità di modulazione

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.

High-throughput Purificazione di affinità-tagged proteine ​​ricombinanti

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 ...