SW e TMV sono supportati dal NCRR e NHLBI presso il National Institutes of Health. BSK e HS sono supportati dal NINDS e NIGMS del National Institutes of Health.

Procedure sperimentali La procedura sperimentale (Fig. 2) si compone di quattro parti che sono descritti in un modo graduale di seguito. Parte 1: subcloning ed espressione del C-terminale del recettore P2X2. Abbiamo espresso tutta la lunghezza C-terminale del recettore P2X2 nei batteri per identificare le proteine ​​del cervello a cui si lega. <ol><li> Il C-terminale (residui 353-472) del recettore P2X2 (Fig. 1) è stato amplificato mediante PCR, clonati in pGEX 4NT1 (GE Life Sciences) e verificato con il sequenziamento. </li><li> Il plasmide ricombinante è stato trasformato in E. Coli (BL21) per l&#39;espressione di proteine ​​ricombinanti. </li><li> Glicerolo scorte di batteri (E. coli BL21) contenente i P2X2 CT-GST plasmide sono stati raschiati con un puntale sterile e inoculata in 5 ml di Luria-Bertani (LB) con idonei mezzi di comunicazione marcatore selettivo (50 g / ml di ampicillina) e incubato per una notte in un agitatore orbitale a 37 ° C. </li><li> Le culture cresciute overnight sono stati inoculati in 250 ml di media LB con un antibiotico adatto (50 mg / ml di ampicillina) e incubato in un agitatore orbitale a 250 rpm per 2-3 ore a 37 ° C fino a quando la densità ottica a 600 nm raggiunto ~ 0,6-0,7. </li><li> La cultura è stata indotta con 1 mM di IPTG e ulteriormente incubate a 37 ° C per 3 ore per l&#39;espressione della proteina. </li><li> La cultura è stato poi girato a 5000 g per 15 min a 4 ° C (1 ml di coltura è stato salvato per analizzare il livello di espressione ed etichettato come &#39;indotto&#39;). Supernatante è stato scartato e il pellet è stato risospeso in 20 ml di Saline Tris-EDTA (STE) buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH 8,0). </li><li> La sospensione è stata filata a 5000 g per 15 minuti in un tubo da 50 ml falco. Il supernatante è stato scartato e la semi-secco pellet è stato congelato a-70C durante la notte. </li><li> I-70C pellet è stato risospeso in 40 ml di tampone di lisi freddo ghiaccio (2% (w / v) sarkosyl, 15 mg di lisozima, 150 mM NaCl, 50 mM Tris pH7.5, 5 mM DTT e completa compressa inibitore della proteasi) , e la sospensione è stata incubata in ghiaccio per 30 min. Durante questa incubazione, 3 ml Glutatione-Sepharose perline 4B sono stati lavati con 20 ml di tampone salino fosfato (PBS) e 20 ml T-PBS (0,1% (v / v) tritonX-100 in PBS), seguita da PBS. Le perle sono stati risospesi in 1 ml di PBS. </li><li> La sospensione batterica è stata freeze-scongelati 3 volte in azoto liquido e sonicato (1s on / off, 30 micron per 3 minuti in ghiaccio) e ulteriormente incubate per 30 minuti con il 4% (v / v) Triton X-100, 10 mM MgSO 4 e 2 mM ATP sul ghiaccio. </li><li> Il lisato è stato girato a 20000 g per 15 minuti a 4C e pellet è stato scartato (un campione di pellet e 1 ml di surnatante è stato salvato per la pagina di SDS per testare la quantità di proteine ​​nel citoplasma e membrane). Il supernatante è stato incubato con le perline per 1 ora (o tutta la notte) a 4C. </li><li> Le perle erano filata a 500 g per 5 minuti e il supernatante è stato scartato (campione è stato salvato per la frazione non legata). Le perle sono stati lavati con T-PBS cinque volte (lavaggi sono stati salvati per i controlli di lavaggio). </li><li> Le perle sono stati poi risospesi in PBS per dare il 50% (w / v) liquami e conservati in frigorifero a 4C fino al momento dell&#39;uso. La concentrazione delle proteine ​​è stato stimato da Bradford test (per le istruzioni del produttore). </li></ol> Parte 2: Preparazione dei lisati intero cervello P2X2 recettori sono noti per essere abbondantemente espresso nel cervello 8. Nella presente analisi, abbiamo cercato di identificare le proteine ​​che interagiscono con i recettori P2X2 da lisati cervello di ratto suo complesso (Fig. 3). <ol><li> Cervello di ratto adulto è stato raccolto (animali sono stati sacrificati in base ad un protocollo approvato IAACUC-UCLA e secondo la guida NIH per la cura e l&#39;uso di animali da laboratorio) e risciacquati immediatamente ghiaccio PBS freddo (3X). </li><li> Per lisare le cellule da 10 ml (~ 5 volte il peso fresco del cervello raccolte) di tampone di lisi freddo ghiaccio contenente 150 mM NaCl, 50 mM Tris-HCl, pH 7,4, 10 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM Na 3 VO 4, 1 mM PMSF, 5 mg / ml di leupeptina, 1% (v / v) NP-40, e un inibitore della proteasi tavoletta cocktail è stato aggiunto al cervello raccolto. </li><li> Tessuto cerebrale è stato omogeneizzato su ghiaccio con un omogeneizzatore Dounce vetro fino a una miscela di consistenza omogenea è stato raggiunto. </li><li> Lisato cellulare è stato girato a 2000 g per 5 minuti per rimuovere le cellule ininterrotta. Il surnatante è stato raccolto e girato nuovamente a 29000 g per 60 minuti per rimuovere organuli intatti e aggregati membrana. </li><li> Immediatamente dopo la centrifugazione il surnatante è stato trasferito in una provetta pulita. La concentrazione proteica del lisato intero cervello è stata misurata mediante saggio Bradford (di solito ~ 15 mg / ml). </li><li> Il lisato è stato aliquotati e conservato a-70C. </li></ol> Parte 3: Isolamento di P2X2 C-coda proteine ​​associate Per identificare i partner legame di P2X2 CT-GST da lisati intero cervello, un pull down test è stato eseguito utilizzando CT-GST immobilizzato su glutatione sefarosio perline 4B come esca. Per i controlli, GST solo legati a microsfere è stato utilizzato. <ol><li> Cervello lisato è stato precleared con perline GST per 1 ora a 4C. Il pellet è stato scartato ed il surnatante è stato utilizzato per ulteriori analisi. </li><li> La stessa quantità di GST e P2X2 CT-GST (10 mcg) legato a glutatione sefarosio 4 perle B sono state incubate overnight con 100 g di proteine ​​precleared solubilizzato da lisato cervello di ratto intero, con rotazione a 4C nel buffer di lisi. </li><li> Perline erano filata a 1000 g per 5 minuti e il supernatante è stato scartato. </li><li> Le perle sono stati lavati una volta con il tampone di lisi e bollito in modificata Laemli tampone [4% (w / v) di SDS, 10% (v / v) 2-mercaptoetanolo, 2% (v / v) glicerolo, 0,004% (w / v) bromofenolo blu e 0,125 M TrisCl pH 6,8] per 5 min. </li><li> I campioni sono stati filata a 5000 g per 5 minuti e il surnatante separato del 10% SDS-PAGE (Fig. 4). Gel sono stati colorati con gel SYPRO proteine ​​Rubino macchia (secondo le istruzioni del produttore) e visualizzati sotto luce ultravioletta. P2X2 CT-proteine ​​associate sono stati confrontati con quelli recuperati utilizzando GST-nullo come esca. Quattro repliche biologiche sono state eseguite (cioè quattro cervelli e quattro bassi tirare indipendenti). </li></ol> Parte 4: Identificazione delle proteine. Le proteine ​​separate mediante elettroforesi su gel sono state escisse dal gel e identificati mediante spettrometria di massa 12. Nota: l&#39;assenza di polvere durante il processo è fondamentale per ridurre la contaminazione cheratina. Lo sperimentatore deve indossare una maschera, retina per capelli e guanti in ogni momento e non toccare mai la regione di interesse gel senza l&#39;uso di guanti. <ol><li> Tutte le bande di proteine ​​visibile recuperato dal menu a discesa P2X2 sono stati asportati con una lametta pulita e tagliata a dadini (Fig. 4). Regioni corrispondenti del gel nel GST-pull down nullo corsia sono stati asportati nello stesso modo senza riguardo alle macchie banda (bande specifiche per la GST-esca nulli sono stati asportati e individuati) per assicurare che la presenza di proteine ​​di sotto del livello di sensibilità colorazione non è stato mancato. </li><li> I pezzi di gel sono stati incubati con 200 ml di 50 mM di bicarbonato di ammonio nel 50% (v / v) acetonitrile (ACN) e dei tubi in agitazione per 15 minuti a temperatura ambiente. Ripetere questa fase di lavaggio. </li><li> I pezzi di gel sono stati disidratati con l&#39;aggiunta di 200 microlitri acetonitrile (pezzi gel dovrebbe restringersi e diventare opaco a colori). </li><li> Acetontrile è stato rimosso ei pezzi gel essiccato in un speedvac per ~ 10 min. </li><li> I pezzi di gel sono state poi incubate con 30 microlitri della preparati al momento 10 mM DTT/10 mM Tris (2-carbossi-etil) cloridrato fosfina (TCEP) del volume sufficiente per immergere i pezzi. Questo è stato lasciato per 30 minuti a bagnomaria 56C. </li><li> Avanti la soluzione digitale terrestre è stato sostituito con 100 l 500 mM di bicarbonato di ammonio in soluzione acetonitrile 50% e le fette di gel lavata con il bicarbonato ammounium. </li><li> La soluzione di lavaggio è stata aspirata e 200 ml di acetonitrile aggiunto a disidratare il gel. </li><li> Acetonitrile è stato poi rimosso e 100 ul preparati al momento 100 soluzione Iodoacetamide mM è stato aggiunto al alchilato sulfhydryls libero. Questo passo ha proceduto per 25 minuti a temperatura ambiente al buio. </li><li> Soluzione Iodoacetamide è stato rimosso ed i pezzi di gel sono stati lavati con 200 ml di bicarbonato di ammonio 50 mM nel 50% acetontrile (pH 8,0) per 2 minuti, mentre vortex. Questa fase di lavaggio è stata ripetuta. </li><li> Dopo aver rimosso la soluzione di lavaggio, i pezzi di gel sono stati incubati con 200 microlitri acetonitrile che è stato poi rimosso da speedvac per ~ 10 minuti. </li><li> A questo punto i pezzi di gel sono pronti per la digestione tripsina. Pezzi di gel sono stati incubati in 30 microlitri soluzione di tripsina (20 ng / mL concentrazione di lavoro) e messo in ghiaccio per 10 min per i gel di essere ben gonfio. Tripsina in eccesso è stato rimosso e reidratato particelle di gel sovrapposto con 30 microlitri con 50 mM di bicarbonato di ammonio per tenerli immersi per tutta la digestione, che è stato permesso di proseguire per 12 a 16 ore a 37 ° C. </li><li> Cinque microlitri del 5% (v / v) di acido formico viene aggiunto per disattivare la digestione tripsina e l&#39;arresto. </li><li> Tubi erano in agitazione per circa 15 minuti e centrifugati brevemente per portare il liquido sul fondo della provetta che è stato poi trasferito in un pulito ed etichettato 0,5 ml Fiala. </li><li> Prossimi 30 ml di 0,1% (v / v) in acido formico 50% acetonitrile è stato aggiunto a pezzi gel quindi erano appena coperto seguito da vortex due volte per 10-15 minuti, separati per centrifugazione breve. Questo passo è stato ripetuto una volta, in sostituzione del acido formico-acetonitrile tra i passaggi. </li><li> Il liquido finale è stato rimosso e tutte le soluzione di estrazione combinati in un singolo flaconcino. Questo volume è stato ridotto a circa il 30 uL nel speedvac. Il campione è ora pronto per l&#39;inversione di fase nano-cromatografia liquida e l&#39;identificazione delle proteine ​​mediante spettrometria di massa. In questo esempio specifico, questo passo è effettuata su Orbi-trappola spettrometro di massa tandem da ThermoFisher (scelta per la sua accuratezza di massa elevata, ciclo rapido per il rilevamento delle proteine ​​e le caratteristiche di funzionamento in generale robusta), anche se altri modelli e strumenti produttori &#39;potrebbe essere facilmente accoppiato con l&#39;approccio descritto a questo punto. </li><li> I dettagli e criteri per l&#39;analisi di spettrometria di massa sono ora brevemente presentati. Tutti i peptidi sono stati separati da fase inversa nano-LC (Eksigent) e peptidi sono stati caricati su una C18 fase inversa colonna ad un flusso di 3 ml / min. Una fase mobile è stata dello 0,1% di acido formico e 2% ACN in acqua; B fase mobile è stata dello 0,1% di acido formico e il 20% d&#39;acqua in ACN. Peptidi sono stati eluiti dalla colonna ad una portata di 220 nl / min con un gradiente lineare B dal 5% al ​​50% B più di 90 minuti, poi al 95% di B in 5 minuti e, infine, mantenendo costante B del 95% per 5 min . Spettri sono stati acquisiti in data-dependent modalità con le Orbi-trappola usati per le scansioni MS e LTQ per MS / MS scansioni. Peptidi sono stati identificati dalla ricerca gli spettri sul database ratto IPI v.3.53 con l&#39;algoritmo SEQUEST integrato nel pacchetto software Bioworks. Ogni peptide soddisfatto i seguenti criteri: XCorr ≥ 2 (+1), ≥ 3 (+2), ≥ 4 (+3), e DeltaCN&gt; 0,1. Tutti gli spettri utilizzati per l&#39;identificazione delle proteine ​​sono stati ispezionati manualmente e non proteine ​​accettato quando a meno di due peptidi sono stati identificati. Proteine ​​presenti solo dopo ciascuna delle quattro P2X2 bassi tirare e assente durante GST-nullo tirare bassi sono stati considerati per ulteriori analisi. </li></ol><img src="/files/ftp_upload/1178/1178fig1.jpg" alt="figura 1" /> Figura 1. Rappresentazione schematica di una subunità del recettore P2X2. R. Il cartone animato illustra la topologia di una subunità del recettore P2X2. Il dominio citosolico è composto da N e C termini. Il C-terminale del recettore P2X2 (rosso) è stato utilizzato come esca per tirare giù saggio. Sequenza di acido B. aminoacidi del recettore P2X2 C-terminale utilizzato in questo studio. <img src="/files/ftp_upload/1178/1178fig2.jpg" alt="" /> Figura 2. Diagramma di flusso e la linea del tempo del protocollo usato per l&#39;espressione, la purificazione, tirare verso il basso e l&#39;identificazione delle proteine. Mostriamo il contorno del protocollo con la linea del tempo. Lisato cervello di ratto adulto è stato preparato fresco immediatamente prima dell&#39;uso per il pull down test. <img src="/files/ftp_upload/1178/1178fig3.jpg" alt="" /> Figura 3. Rappresentazione schematica delle analisi proteomica binario di P2X2 C-terminale della rete nel cervello di ratto. Il C-terminale del recettore P2X2 fuse con le proteine ​​GST legati a microsfere GST è stato utilizzato per abbattere i test. Cervello lisato è stato preparato e proteine ​​sono state incubate con le proteine ​​ricombinanti immobilizzato. Frazione libera è stato lavato con il tampone di lisi. Le proteine ​​sono stati identificati mediante spettrometria di massa. <img src="/files/ftp_upload/1178/Fig%204%20Singh%20et%20al.jpg" alt="" /> Figura 4. Identificazione di partners vincolante del C-terminale del recettore P2X2. Sypro gel colorato che mostra uno spettro di proteine ​​putative che interagiscono con il C-terminale dei recettori fuse con GST (blue box). Corsie controlli per GST solo (scatola gialla) e glutatione perline sefarosio solo sono mostrati. Le frecce indicano esempi di gruppi unici che sono stati asportati per ulteriori analisi mediante spettrometria di massa.

<ol>
	<li>Hille, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion channels of excitable membranes. , (2001).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+P2X+receptors+and+ATP+signalling+at+synapses.">Molecular physiology of P2X receptors and ATP signalling at synapses.</a> Nat Rev Neurosci. 2, 165-174 (2001).</li><li>Egan, T. M., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+calcium+ions+to+P2X+channel+responses.">Contribution of calcium ions to P2X channel responses.</a> J Neurosci. 24, 3413-3420 (2004).</li><li>Burnashev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+permeability+of+ligand-gated+channels.">Calcium permeability of ligand-gated channels.</a> Cell Calcium. 24, 325-332 (1998).</li><li>Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling.">Calcium signaling.</a> Cell. 131, 1047-1058 (2007).</li><li>Levitan, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+protein+complexes+associated+with+neuronal+ion+channels.">Signaling protein complexes associated with neuronal ion channels.</a> Nat Neurosci. 9, 305-310 (2006).</li><li>Khakh, B. S., North, 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=P2X+receptors+as+cell-surface+ATP+sensors+in+health+and+disease.">P2X receptors as cell-surface ATP sensors in health and disease.</a> Nature. 442, 527-532 (2006).</li><li>Roger, S., Pelegrin, P., Surprenant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+P2X7+receptor+currents+and+membrane+blebbing+via+constitutive+and+dynamic+calmodulin+binding.">Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding.</a> J Neurosci. 28, 6393-6401 (2008).</li><li>North, 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=Molecular+physiology+of+P2X+receptors.">Molecular physiology of P2X receptors.</a> Physiol Rev. 82, 1013-1067 (2002).</li><li>Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+union+of+pharmacology.+XXIV.+Current+status+of+the+nomenclature+and+properties+of+P2X+receptors+and+their+subunits..">International union of pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits..</a> Pharmacol Rev. 53, 107-118 (2001).</li><li>Young, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+shape,+architecture+and+size+of+P2X4+receptors+determined+using+fluorescence+resonance+energy+transfer+and+electron+microscopy.">Molecular shape, architecture and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.</a> J Biol Chem. 283, 26241-26251 (2008).</li><li>Edmondson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+epsilon+signaling+complexes+include+metabolism-+and+transcription/translation-related+proteins:+complimentary+separation+techniques+with+LC/MS/MS.">Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS.</a> Mol Cell Proteomics. 1, 421-433 (2002).</li><li>Evans, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthosteric+and+allosteric+binding+sites+of+P2X+receptors.">Orthosteric and allosteric binding sites of P2X receptors.</a> Eur Biophys J. 38, 319-327 (2009).</li></ol>

Canali ionici sono una classe importante di proteine ​​integrali di membrana. Essi contengono pori riempiti d&#39;acqua che selettivamente permettono il movimento degli ioni i loro gradienti elettrochimici attraverso la membrana plasmatica. Ion cancello canali tra gli stati aperti e chiusi. Il passo gating è innescato da trasmettitori (es. ATP) in caso di P2X canali ionici ligando gated, o può essere regolata da interazioni con altre proteine. L&#39;ultimo decennio ha visto un aumento della nostra comprensione di ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Acetonitrile</td>
<td>Reagent</td>
<td>JT Baker</td>
<td>9829-02</td>
<td>&nbsp;</td>
<tr>
<td>Acrylamide</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0156</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1507-01</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
<tr>
<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotritol (DTT)</td>
<td>Reagent</td>
<td>EMD</td>
<td>3860</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1474-01</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene Glycol tetraacetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
<td>&nbsp;</td>
<tr>
<td>Formic acid</td>
<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione Sepharose 4B beads</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-5132-01</td>
<td>&nbsp;</td>
<tr>
<td>Hydrochloric acid (HCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1758</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>15502</td>
<td>&nbsp;</td>
<tr>
<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I1149</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani (LB) Media</td>
<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L8511</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>Reagent</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Sulphate (MgSO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride (NaCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Flouride (NaF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Orthovanadate (Na3VO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P40</td>
<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
<td>&nbsp;</td>
<tr>
<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
<tr>
<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>170-3125</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N',N'-Tetramethylethylenediamine (TEMED)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9281</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

Ligando-canali ionici alla base della comunicazione sinaptica nel sistema nervoso 1. Nei mammiferi ci sono tre famiglie di canali ligando-dipendenti: il ciclo Cys, il glutammato-dipendenti ed i canali recettori P2X 2. In ogni caso vincolante del trasmettitore porta alla apertura di un poro attraverso il quale gli ioni scendono i loro gradienti elettrochimici. Molti canali ligando-dipendenti sono permeabili agli ioni calcio 3, 4, che hanno ruoli di segnalazione a valle 5 (regolazione dei geni ad esempio) che possono superare la durata di apertura del canale. Così ligando-dipendenti canali in grado di segnalare su scale temporali di ampia portata da pochi millisecondi a giorni. Alla luce di questi importanti ruoli, è necessario capire come ligando-canali ionici si sono regolati da proteine, e come queste proteine ​​possono regolare segnalazione. Studi recenti suggeriscono che molti, se non tutti, i canali possono far parte di complessi proteina di segnalazione 6. In questo articolo spiegheremo come identificare le proteine ​​che si legano al C-terminale aspetti del settore P2X2 recettore citosolico. Recettori P2X sono ATP-dipendenti canali di comunicazione e composto da sette subunità (P2X1-P2X7). Recettori P2X sono ampiamente espresse nel cervello, dove mediano la trasmissione sinaptica eccitatoria e facilitazione presinaptica di neurotrasmettitori versione 7. Recettori P2X si trovano nelle cellule eccitabili e non eccitabili e mediare ruoli chiave nella segnalazione neuronale, l&#39;infiammazione e la funzione cardiovascolare 8. P2X2 recettori sono abbondanti nel sistema nervoso 9 e sono al centro di questo studio. Ogni subunità P2X è pensato di possedere due segmenti di membrana spanning (TM1 e TM2) separate da una regione extracellulare 7 e intracellulare N e C Termini (Fig. 1a) 7. Subunità P2X 10 (P2X1-P2X7) mostrano omologia di sequenza del 30-50% a livello di aminoacidi 11. Recettori P2X contengono solo tre subunità, che è il più semplice stechiometria tra recettori ionotropici. Il P2X2 C-terminale è composta da 120 aminoacidi (Fig. 1b) e contiene numerose proteine ​​siti consenso docking, supportando l&#39;ipotesi che P2X2 recettore può essere parte di complessi di segnalazione. Tuttavia, anche se diverse funzioni sono state attribuite al C-terminale del recettore P2X2 9 nessuno studio ha descritto i partner molecolari che paio al lato di questa proteina intracellulare attraverso tutta la lunghezza C-terminale. In questo documento si descrivono i metodi di un approccio di proteomica per identificare le proteine ​​che interagiscono con l&#39;intera lunghezza C-terminale del recettore P2X2.

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Descriviamo un semplice protocollo per identificare le proteine ​​del cervello che si legano al capolinea pieno C lunghezza di ATP-dipendenti P2X2 recettori. L&#39;estensione e l&#39;applicazione sistematica di questo approccio a tutti i recettori P2X dovrebbe portare ad una migliore comprensione di segnalazione dei recettori P2X.

Proteomica per identificare proteine ​​che interagiscono con P2X2 Ligand-Gated canali di comunicazione

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

proteomics-to-identify-proteins-interacting-with-p2x2-ligand-gated

Research

JoVE Journal

Biology

14.6K Views.  David Geffen School of Medicine, University of California, Los Angeles. Descriviamo un semplice protocollo per identificare le proteine ​​del cervello che si legano al capolinea pieno C lunghezza di ATP-dipendenti P2X2 recettori. L'estensione e l'applicazione sistematica di questo approccio a tutti i recettori P2X dovrebbe portare ad una migliore comprensione di segnalazione dei recettori P2X.

Video: Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

L&#39;identificazione di complessi proteici con la proteomica quantitativa in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Ricerca

Biologia

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - un metodo per identificare trascrittoma livello i siti di legame delle proteine ​​RNA binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Utilizzando SCOPE per identificare potenziali Motivi regolamentari in geni Coregulated

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

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 ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Cristallizzazione high-throughput di proteine ​​di membrana con il metodo lipidica Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificato lievito-sistema del doppio ibrido per identificare proteine ​​che interagiscono con il fattore di crescita progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Reverse lievito sistema Two-ibrida per identificare mammiferi Nuclear Receptor residui che interagiscono con leganti e / o antagonisti

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Bottom-up e Shotgun Proteomica per identificare una completa Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Glicopeptide Capture per Proteomica cellulari di superficie

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Proteomica per identificare proteine ​​che interagiscono con P2X2 Ligand-Gated canali di comunicazione

Proteomica per identificare proteine che interagiscono con P2X2 Ligand-Gated canali di comunicazione