Questo lavoro è stato finanziato dalla Energy Biosciences Institute concedere OO0G01.

Il metodo OLIMP è esemplificata sulla emicellulosa più importanti presenti nelle pareti cellulari delle piante dicot, xyloglucan, utilizzando un endoglucanase come glycosylhydrolase. Il metodo è dimostrato con tutta piantine di Arabidopsis come fonte di tessuto vegetale. Materiale della matrice extracellulare di enzimi e può essere sostituito in base all&#39;analisi desiderato utilizzando la stessa procedura. 1. Isolamento della parete cellulare <ol><li> Harvest cinque 5 giorni di età le piante intere Arabidopsis o l&#39;equivalente del materiale desiderato e trasferirli in una provetta di reazione 1,5 ml. Assicurarsi che il materiale si trova nel fondo della provetta. </li><li> Aggiungere due sfere di metallo tre millimetri al tubo di reazione sulla parte superiore del campione e snap-congelare in azoto liquido. </li><li> Frantumare il campione congelato utilizzando un mulini a palle (2:30 min, 25 Hz). </li><li> Aggiungere 1 ml di etanolo al 70% acquosa al campione. Rimuovere le sfere di metallo con un magnete. Vortex accuratamente. </li><li> Centrifugare campione a 14.000 rpm per 10 minuti per far sedimentare il residuo insolubile alcool contenente il materiale della parete cellulare. </li><li> Rimuovere con cautela il surnatante tramite aspirazione. Fare attenzione a non disturbare il pellet. </li><li> Aggiungere 1 ml di cloroformio / metanolo (1:1 v / v). Vortex a fondo per risospendere il pellet. </li><li> Centrifugare campione a 14.000 rpm per 10 minuti e rimuovere con attenzione il surnatante tramite aspirazione. Fare attenzione a non disturbare il pellet. </li><li> Secco il campione con un concentratore. </li></ol> 2. Solubilizzazione di oligosaccaridi <ol><li> Per la solubilizzazione del polisaccaride particolare forma dei suoi oligosaccaridi risospendere il pellet in secco 25μl di un buffer adeguato. Per la formiato endoglucanase ammonio 50 mM, pH4.5, viene utilizzato. Aggiungi 0.2U di endoglucanase. Vortex la sospensione e liquido spin down. </li><li> Incubare il campione per 16h a 37 ° C in un incubatore, agitazione a 300rpm. </li><li> Togliere il solvente del digest (acqua) con un concentratore. </li></ol> 3. MALDI-TOF analisi del oligosaccaridi rilasciato <ol><li> BioRex MSZ 501 perle di resina a scambio cationico sono utilizzati per eliminare i sali ed enzimi dal campione digerito. Condizione le perle inserendo una aliquota in una colonna vuota e lavare le perline con abbondante acqua. </li><li> Aggiungi 5-10 perline BioRex al campione digerito e asciugato. Quindi aggiungere acqua 10μl, per piccole quantità di materiale 5μl di acqua può essere utilizzata. Immergere le perline nella soluzione con una breve rotazione del tubo. Incubare per almeno 10 minuti a temperatura ambiente. </li><li> Spot 2μl matrice (2,5-dihydroxybenzoic acido (DHB), 10mg/ml in acqua) su una piastra MALDI. Far evaporare il solvente sotto vuoto matrice porta a cristalli omogenea matrice chimica. </li><li> 2μl posto della soluzione del campione dissalati in cima alla matrice di cristalli sulla piastra bersaglio. Se si vuole individuare un gran numero di campioni solo continuare a spotting per un massimo di 3 minuti. Questo lasso di tempo assicura che il primo campione individuato non è ancora asciutto. Aspettare un 2min aggiuntivo per assicurare una miscelazione di ri-dissolto matrice e le molecole del campione oligosaccaridi nel campione ultimi maculato. Poi asciugare la piastra bersaglio sotto vuoto. Il campione / matrice mix dovrebbe cristallizzare in meno di 1 minuto per permettere cristallizzazione omogenea. </li><li> Mettere in una piastra segnale MALDI-TOF spettrometro di massa. La macchina deve essere impostata in modalità positiva reflectron con una tensione di accelerazione di 20000V e un ritardo di 350 nm, il range di massa selezionato dovrebbe essere 500-3000 Da (può cambiare dipende dalla oligomeri previsto). Iniziare a sparare il laser e raccogliere 100-200 spettri per campione, che sono compilati per generare uno spettro rappresentativo medio (Figura 2A). Ioni che rappresentano oligosaccaridi specifici possono essere identificati mediante il loro peso su carica (m / z) rapporto. Le intensità di ioni (altezza di picco) di tutti gli ioni di interesse deve essere aggiunto fino al 100% con conseguente abbondanza relativa di ogni oligosaccaridi del campione (Figura 2B). </li></ol> 4. Nell&#39;analisi OLIMP situ OLIMP può essere utilizzato anche direttamente sul tessuto tralasciando alcuna iniziativa preparazione muro. Come esempio eziolata hypocotyls di Arabidopsis come fonte tessuto vegetale sono utilizzati. Ancora una volta, un endoglucanase viene utilizzato per determinare la struttura del xyloglucan emicellulose. Enzima e il materiale del tessuto possono essere sostituiti in base all&#39;analisi desiderato utilizzando la stessa procedura. <ol><li> Harvest uno pianticella eziolata e collocarla su una piastra MALDI vuoto e lasciare che la piantina secca. </li><li> Aggiungi 0.5μl endoglucanase (0.4U/μl, disciolto in formiato di ammonio 50 mM, pH4.5) sulla piantina essiccata nella posizione desiderata. Assicurarsi che la goccia enzima tocca in parte la piastra bersaglio. </li><li> Posizionare la piastra segnale MALDI in un contenitore chiuso con umidità saturazione per evitare che la caduta di enzima si asciuga. Incubare la piastra nella camera per 16h a 37 ° C. </li><li> Asciugare la piastra MALDI con il tessuto della pianta e la caduta di enzima sotto vuoto. </li><li> Aggiungi 0.5μl matrice (DHB; 10 mg / l) in cima ad ogni punto enzima secca. Lasciate riposare per 2 minuti. </li><li> Asciugare la piastra segnale MALDI sotto vuoto. </li><li> Analizzare campioni con il MALDI-TOF. Luogo piastra con il tessuto e l&#39;enzima / matrice spot (s) in un MALDI-TOF spettrometro di massa. La macchina deve essere impostata in modalità positiva reflectron con una tensione di accelerazione di 20000V e un ritardo di 350 nm, il range di massa selezionato dovrebbe essere 500-3000 Da (può cambiare dipende dalla oligomeri previsto). </li><li> Iniziare a sparare il laser sulla matrice / campione cristalli NEXT per il tessuto. Assicuratevi di non colpire il tessuto stesso, come in questo caso il tempo di volo delle molecole sarà spento. Raccogliere intorno 20-50 spettri per punto, che sono compilati per generare uno spettro rappresentativo medio (Figura 3). Ioni che rappresentano oligosaccaridi specifici possono essere identificati mediante il loro peso su carica (m / z) rapporto. Le intensità di ioni (ioni di altezza) possono essere riportati e tutti gli ioni di interesse deve essere aggiunto fino al 100% con conseguente abbondanza relativa di ogni oligosaccaridi nel campione. </li></ol> 5. Rappresentante Risultati Un esempio di analisi OLIMP del xyloglucan emicellulose presenti in piantine di Arabidopsis è illustrato nella figura 2. A causa delle differenze di massa degli ioni e l&#39;enzima noto ben caratterizzato (endoglucanase) specificità gli ioni possono essere assegnati a specifiche strutture oligosaccaridi (Figura 2A). Ovviamente, isomeri strutturali non possono essere distinte. L&#39;assunto di base per la determinazione della relativa abbondanza dei vari oligosaccaridi (Figura 2B) è che la loro spec fattore di risposta di massa è molto simile per quelli oligosaccaridi. Come mostrato qui, la quantificazione OLIMP è altamente riproducibile. Tuttavia, si ricorda che la robustezza del metodo dipende in larga misura il segnale ai rapporti rumore dei vari ioni oligosaccaridi. Ad esempio la contaminazione con sali o minori quantità di oligosaccaridi può diminuire tale rapporto. L&#39;analisi OLIMP sullo stesso tessuto (analisi in situ) consente lo studio di aree molto piccole e definito e prevede la preparazione del campione molto poco. Nell&#39;esempio qui presentato (Figura 3) non qualitativa (ioni stesso), ma differenze quantitative (variazione di intensità di ioni) sono state osservate tra le riprese e la radice dei tessuti della piantina Arabidopsis. Permutazioni della OLIMP-metodo potrebbe causare così il tessuto &quot;imaging&quot;. <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="Figura 1" /> Figura 1. Diagramma di flusso della procedura OLIMP utilizzando piantine di Arabidopsis tutto come fonte vegetale. In primo luogo, il tessuto è macerata e pareti di materiale cellulare è preparato (immagine modificata da Fujino et al. 10). Poi oligosaccaridi di un polisaccaride muro particolare vengono rilasciati utilizzando una idrolasi specifica. Infine, le abbondanze relative delle oligosaccaridi solubili sono determinati utilizzando MALDI-TOF spettrometria di massa. <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="Figura 2" /> Figura 2. Abbondanza relativa di oligosaccaridi xyloglucan in piantine di Arabidopsis eziolata come determinato dal OLIMP. A) Rappresentante spettro di massa oligosaccaride xyloglucan; ogni ione rappresenta una struttura specifica oligosaccaridi, isomeri strutturali non possono essere distinte. B) diagramma a barre corrispondenti per la determinazione della relativa abbondanza oligosaccaridi. <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="Figura 3" /> Figura 3. Nell&#39;analisi OLIMP situ utilizzando piantine di Arabidopsis eziolata come esempio. Ogni enzima / digestione goccia (cerchi colorati) possono essere analizzati in modo indipendente e spettri di massa corrispondente possono essere ottenuti e analizzati. <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="Figura 4" /> Figura 4. Spettro OLIMP del xilano emicellulosa ottenuto da digerire materiale fogliare Miscanthus con un xilanasi (Megazyme).

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+xylan+synthesis+mutants+reveals+new+insight+into+the+mechanisms+of+xylan+synthesis.">Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis.</a> Plant Journal. 52 (6), 1154-1154 (2007).</li><li>Lee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARVUS+gene+is+expressed+in+cells+undergoing+secondary+wall+thickening+and+is+essential+for+glucuronoxylan+biosynthesis.">The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis.</a> Plant Cell Physiol. 48 (12), 1659-1659 (2007).</li><li>Obel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectin+may+hinder+the+unfolding+of+xyloglucan+chains+during+cell+deformation:+implications+of+the+mechanical+performance+of+Arabidopsis+hypocotyls+with+pectin+alterations.">Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations.</a> Molecular Plant . , (2009).</li><li>Cavalier, D. M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+xyloglucan+xylosyltransferases+catalyze+the+addition+of+multiple+xylosyl+residues+to+cellohexaose.">Two xyloglucan xylosyltransferases catalyze the addition of multiple xylosyl residues to cellohexaose.</a> Journal of Biological Chemistry. 281 (45), 34197-34197 (2006).</li><li>Leonard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+Arabidopsis+gene+encoding+a+GH95+alpha1,2-fucosidase+active+on+xyloglucan+oligo-+and+polysaccharides.">Identification of an Arabidopsis gene encoding a GH95 alpha1,2-fucosidase active on xyloglucan oligo- and polysaccharides.</a> Phytochemistry. 69 (10), 1983-1983 (2008).</li><li>Cavalier, D. M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+xyloglucan+xylosyltransferases+catalyze+the+addition+of+multiple+xylosyl+residues+to+cellohexaose.">Two xyloglucan xylosyltransferases catalyze the addition of multiple xylosyl residues to cellohexaose.</a> Journal of Biological Chemistry. 281 (45), 34197-34197 (2006).</li><li>Lee, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+irregular+xylem9+mutant+is+deficient+in+xylan+xylosyltransferase+activity.">The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity.</a> Plant and Cell Physiology. 48 (11), 1624-1624 (2007).</li><li>Iglesias, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+glycosidases+active+against+xyloglucan+oligosaccharides+of+Arabidopsis+thaliana.">Apoplastic glycosidases active against xyloglucan oligosaccharides of Arabidopsis thaliana.</a> Plant and Cell Physiology. 47 (1), 55-55 (2006).</li><li>Fujino, T., Sone, Y., Mitsuishi, Y., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cross-links+between+cellulose+microfibrils,+and+their+occurrence+during+elongation+growth+in+pea+epicotyl.">Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl.</a> Plant Cell Physiol. 41 (4), 486-486 (2000).</li><li>Vanzin, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mur2+mutant+of+Arabidopsis+thaliana+lacks+fucosylated+xyloglucan+because+of+a+lesion+in+fucosyltransferase+AtFUT1.">The mur2 mutant of Arabidopsis thaliana lacks fucosylated xyloglucan because of a lesion in fucosyltransferase AtFUT1.</a> PNAS. 99 (5), 3340-3340 (2002).</li><li>Cavalier, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+two+Arabidopsis+thaliana+xylosyltransferase+genes+results+in+plants+deficient+in+xyloglucan,+a+major+primary+cell+wall+component.">Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component.</a> Plant Cell. 20 (6), 1519-1519 (2008).</li><li>Hilz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+liquid+chromatography,+capillary+electrophoresis,+and+mass+spectrometry+methods+to+determine+xyloglucan+structures+in+black+currants.">A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants.</a> Journal of Chromatography A. 1133 (1-2), 275-275 (2006).</li><li>Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+mur1+mutation+on+xyloglucans+produced+by+suspension-cultured+Arabidopsis+thaliana+cells.">Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells.</a> Planta. 214 (1), 67-67 (2001).</li></ol>

Nessun conflitto di interessi dichiarati.

Il metodo OLIMP qui presentata consente una analisi molto sensibile e rapida dei polimeri presenti nelle matrici extracellulari. OLIMP combina il rilascio enzimatico di oligomeri con successiva analisi MALDI-TOF. La generazione di un MALDI-TOF spettro richiede meno di un minuto, quindi OLIMP è adatto per una vasta gamma di applicazioni, tra high-throughput studi come schermi mutante. OLIMP non si limita ai polisaccaridi vegetali, ma può potenzialmente essere applicato a una vasta gamma di polimeri, limitato solo dalla disponibilità di specifici enzimi idrolitici. Tuttavia, una limitazione di OLIMP è che l&#39;abbondanza assoluta del polimero non può essere ottenuto. Come accennato prima OLIMP può essere usato per studiare la struttura di una varietà di polisaccaridi presenti nella matrice extracellulare di una diversità di specie. A titolo di esempio, la Figura 4 rappresenta uno spettro OLIMP della emicellulosa principali specie di erba, xilano. Qui, parete materiale cellulari derivate da l&#39;erba Miscanthus temperato è stato digerito con un nasi.

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		<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>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

oligo mass profiling (olimp) of extracellular polysaccharides

Il contatto diretto delle cellule con l&#39;ambiente è mediato in molti organismi da una matrice extracellulare. Un aspetto comune delle matrici extracellulari è che essi contengono frazioni di zuccheri complessi in forma di glicoproteine, proteoglicani, e / o polisaccaridi. Alcuni esempi sono la matrice extracellulare degli esseri umani e cellule animali che consistono principalmente di proteine ​​fibrillari e proteoglicani o la base di polisaccaridi pareti delle cellule delle piante e funghi, e il proteoglicani / glicolipide pareti cellulari a base di batteri. Tutti questi glycostructures giocano un ruolo fondamentale nella cellula-cellula e comunicazione cellula-ambiente e di segnalazione. Un esempio straordinario complesso di una matrice extracellulare è presente nelle pareti delle cellule delle piante superiori. Il loro muro è fatto quasi interamente di zuccheri, fino al 75% del peso secco, e consiste nella più abbondanti biopolimeri presenti su questo pianeta. Pertanto, la ricerca è condotta come utilizzare questi materiali migliori come carbon-neutral delle risorse rinnovabili in sostituzione di prodotti petrolchimici derivati ​​da combustibili fossili. La sfida principale per la conversione del carburante rimane la riluttanza dei muri per degradazione enzimatica o chimica a causa della glycostructures unici presenti in questo biocomposite unico. Qui, vi presentiamo un metodo per l&#39;analisi rapida e sensibile di glycostructures cellula vegetale muro. Questo metodo di analisi di massa oligo (OLIMP) si basa il rilascio enzimatica di oligosaccaridi da facilitare glycosylhydrolases Pareti di materiali specifici e successiva analisi delle miscele solubilizzato oligosaccaridi utilizzando matrice laser assistita desorbimento / ionizzazione tempo di volo spettrometria di massa (MALDI-TOF/MS ) 1 (Figura 1). OLIMP richiede pareti delle celle solo 5000 per una analisi completa, può essere effettuata sul tessuto stesso 2, ed è suscettibile di high-throughput analisi 3. Mentre la quantità assoluta dei oligosaccaridi solubilizzato non può essere determinata da OLIMP l&#39;abbondanza relativa degli ioni vari oligosaccaridi possono essere delineate dagli spettri di massa dando intuizioni circa la sostituzione-pattern del polisaccaride nativo presente nel muro. OLIMP può essere utilizzato per analizzare una vasta gamma di polimeri muro, limitato solo dalla disponibilità di enzimi specifici 4. Ad esempio, per l&#39;analisi dei polimeri presenti nei enzimi vegetali della parete cellulare sono disponibili per analizzare i xyloglucan emicellulose utilizzando un xyloglucanase 5, 11, 12, 13, xilano utilizzando un endo-β-(1-4) xilanasi 6,7 , o per polisaccaridi pectiche utilizzando una combinazione di una poligalatturonasi e un methylesterase 8. Inoltre, utilizzando gli stessi principi di OLIMP glycosylhydrolase e anche attività glicosiltransferasi possono essere monitorati e determinato 9.

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

Un modo rapido per guadagnare è descritto lo studio della struttura di polisaccaridi in una matrice extracellulare. Il metodo sfrutta la specificità del glycosylhydrolases e la sensibilità della spettrometria di massa permette piccole quantità di materiale da analizzare. Questa tecnica è adattabile per essere utilizzato direttamente sul tessuto stesso.

Oligo Profiling di massa (OLIMP) di polisaccaridi extracellulari

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K Views.  University of California, Berkeley. Un modo rapido per guadagnare è descritto lo studio della struttura di polisaccaridi in una matrice extracellulare. Il metodo sfrutta la specificità del glycosylhydrolases e la sensibilità della spettrometria di massa permette piccole quantità di materiale da analizzare. Questa tecnica è adattabile per essere utilizzato direttamente sul tessuto stesso.

Video: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Profiling anticorpi Systems Immunoprecipitazione luciferasi (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profilatura di metiltransferasi e altri S-Adenosil- L-Omocisteina-binding proteine ​​mediante spettrometria di massa Compound Capture (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profiling Glycan dei polimeri cellula vegetale da parete usando i microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Analisi della traduzione innesco durante condizioni di stress da polisoma Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Valutazione della Selective mRNA Traduzione in cellule di mammifero per polisoma Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Arricchimento della matrice extracellulare proteine ​​da tessuti e digestione in Peptidi per l&#39;analisi Spettrometria di Massa

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Profilatura cellulare Redox usando la microscopia ad alto contenuto

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...