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>
	<li>Lerouxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+structural+phenotyping+of+plant+cell+wall+mutants+by+enzymatic+oligosaccharide+fingerprinting.">Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting.</a> Plant Physiology. 130 (4), 1754-1754 (2002).</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=Microanalysis+of+plant+cell+wall+polysaccharides.">Microanalysis of plant cell wall polysaccharides.</a> Molecular Plant. 2 (5), 922-922 (2009).</li><li>Mouille, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+analysis+of+primary+cell+wall+composition+in+Arabidopsis.">Quantitative trait loci analysis of primary cell wall composition in Arabidopsis.</a> Plant Physiology. 141 (3), 1035-1035 (2006).</li><li>Bauer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+suite+of+polysaccharide-degrading+enzymes+for+analyzing+plant+cell+walls.">Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls.</a> PNAS. 103 (30), 11417-11417 (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=A+xyloglucan-specific+endo-beta-1,4-glucanase+from+Aspergillus+aculeatus:+expression+cloning+in+yeast,+purification+and+characterization+of+the+recombinant+enzyme.">A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme.</a> Glycobiology. 9 (1), 93-93 (1999).</li><li>Aboughe-Angone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+carbohydrates+from+fruit+pulp+of+Argania+spinosa:+structural+analysis+of+pectin+and+xyloglucan+polysaccharides.">Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides.</a> Carbohydr Res. 343 (1), 67-67 (2008).</li><li>Brown, 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=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.

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

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

Universidad de Cartagena UDEC

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

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Miglioramento in gel riduttive solfo-glycomics β-eliminazione completa per O-linked e di spettrometria di massa

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Ricerca

Chimica

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Glycoproteomics della matrice extracellulare: Un metodo per l&#39;analisi Intact Glycopeptide Utilizzando la spettrometria di massa

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Biochimica

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

Caratterizzazione strutturale di Mannan polisaccaridi di parete cellulare nelle piante usando passo

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Il sequenziamento di Plant parete Heteroxylans Utilizzando enzimatica, chimica (metilazione) e (Spettrometria di Massa, Risonanza Magnetica Nucleare) tecniche fisiche

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Spettrometria di massa, di antigeni glicosfingolipide

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunologia e infezioni

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Guardando verso l'esterno: Isolamento dei polimeri e delle proteine di carboidrati rilasciati in cianobatteria

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Profilazione dei polisomi senza generatori di gradienti o sistemi di frazionamento

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

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 ...

Biologia

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Oligo Profiling di massa (OLIMP) di polisaccaridi extracellulari

Riepilogo

Esplora Altri Video

Capitoli in questo video

Miglioramento in gel riduttive solfo-glycomics β-eliminazione completa per O-linked e di spettrometria di massa

Glycoproteomics della matrice extracellulare: Un metodo per l'analisi Intact Glycopeptide Utilizzando la spettrometria di massa

Caratterizzazione strutturale di Mannan polisaccaridi di parete cellulare nelle piante usando passo

Il sequenziamento di Plant parete Heteroxylans Utilizzando enzimatica, chimica (metilazione) e (Spettrometria di Massa, Risonanza Magnetica Nucleare) tecniche fisiche

Spettrometria di massa, di antigeni glicosfingolipide

Guardando verso l'esterno: Isolamento dei polimeri e delle proteine di carboidrati rilasciati in cianobatteria

Profilazione dei polisomi senza generatori di gradienti o sistemi di frazionamento

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Profiling Glycan dei polimeri cellula vegetale da parete usando i microarrays

Automatizzata Modular High Throughput Screening esopolisaccaride piattaforma Accoppiato con altamente sensibile Analisi Carboidrati impronte digitali