Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
Uploaded: 2018-06-25T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Questo lavoro è stato supportato in parte dalla Natural Science Foundation of China (concedere numeri 31471703, A0201300537 e 31671854 a J.V. e L.L., concedere il numero 31470435 a G.Y.) e il piano di talenti stranieri 100 (concessione numero JSB2014012 di J.V.).

1. raccolta del campione
<ol><li>Acquistare diverse cultivar di fresco ravanello (Raphanus sativus L.).</li>
</ol>2. isolamento della proteina dal ravanello
<ol><li>Omogeneizzare circa 100 g di ravanello fresco con un frullatore di cucina per 10 min.</li>
	<li>Trasferire l'impasto per un tubo di centrifuga da 50 mL e centrifugare a 14.000 × g a 4 ° C per 20 minuti rimuovere il materiale insolubile.</li>
	<li>Trasferire il surnatante con attenzione in una nuova provetta da centrifuga 50 mL e aggiungere un volume equivalente di soluzione di acido tricloroacetico (TCA) di 2 M. Nota: L'aggiunta di TCA precipiterà solubile (glico) proteine.</li>
	<li>Centrifugare a 14.000 × g a 4 ° C per 30 min e rimuovere il surnatante da pellettato (glico) proteine.</li>
	<li>Lavare la pallina con 20 mL di acqua deionizzata e centrifugare a 14.000 × g a 4 ° C per 5 min rimuovere polisaccaridi e oligosaccaridi solubili.</li>
	<li>Ripetere il punto 2.5. quattro volte.</li>
	<li>Risospendere il pellet in 1 mL di acqua deionizzata e trasferimento in una provetta da centrifuga fresca 1,5 mL.</li>
</ol>3. preparazione di N- glicani
<ol><li>Mix 50 µ l della soluzione di proteina dal punto 2.7. (pari a 5 g di ravanello fresco) con 0,2 mU di ricombinante PNGasi H+ in acido acetico 10 mM.</li>
	<li>Incubare a 37 ° C per 12 h, la miscela di reazione. Dopo l'incubazione, centrifugare a 14.000 × g per 5 min rimuovere le proteine extra e degli enzimi.</li>
	<li>Trasferire il surnatante in una provetta da centrifuga fresca 1,5 mL.</li>
</ol>4. purificazione di N- glicani
<ol><li>Preparare la colonna di estrazione in fase solida (SPE) per arricchire gli N- glicani e rimuovere i sali ed altre impurità dalla miscela di reazione, aumentare la selettività dell'agente d'etichettatura fluorogenic 2-aminobenzamide (2-AB) per N- glycan derivatizzazione.<ol>
<li>Aggiungere 3 mL di acqua deionizzata per lavare la colonna.</li>
		<li>Aggiungere 3 mL di soluzione di acetonitrile 80% contenente acido trifluoroacetico (TFA, 0.1% v/v) in attivo la SPE-colonna.</li>
		<li>Aggiungere 3 mL di acqua deionizzata per equilibrare la SPE-colonna.</li>
	</ol>
</li>
	<li>Trasferire il campione dal punto 3.3 sulla colonna e scartare il flusso continuo.</li>
	<li>Aggiungere 1,5 mL di acqua deionizzata per lavare la colonna e scartare il flusso continuo.</li>
	<li>Eluire il N- glicani rilasciato dalla colonna utilizzando 1,5 mL di soluzione acquosa al 20% acetonitrile, contenente 0,1% TFA (v/v) in una provetta da centrifuga da 2 mL.</li>
	<li>Rimuovere il solvente per evaporazione centrifuga a temperatura ambiente. Evaporare fino a quando il campione è completamente asciutto.</li>
</ol>5. fluorescenza derivatizzazione di N- glicani
<ol><li>Preparare 1 mL di soluzione 2-AB, composto da 35 mM 2-AB e 0,1 M cianoboroidruro di sodio in soluzione di acido acetico/solfossido dimetilico (7:3, v/v).</li>
	<li>Aggiungere 5 µ l di soluzione di 2-AB ai campioni secchi (passo 4.5.) derivati da sei differenti ravanelli. Vortex ciascun campione fino a quando completamente sciolto.</li>
	<li>Incubare la miscela per 2 ore a 65 ° C.</li>
	<li>Lasciate raffreddare il campione a temperatura ambiente per 5 minuti e aggiungere 5 µ l di acqua deionizzata e 40 µ l di acetonitrile. Centrifugare le provette a 14.000 x g per 3 min.</li>
	<li>Trasferire 48 µ l dal surnatante in un flaconcino HPLC di 300 µ l alta-recupero. Memorizzare i derivatizzato N- glycan campioni a-20 ° C fino ad un mese.</li>
</ol>6. HILIC-UPLC profilatura di N- glicani
<ol><li>Analizzare i campioni utilizzando un sistema UPLC standard collegato a un detectorset di fluorescenza online alle lunghezze d'onda di eccitazione/emissione della 330 nm/420 nm, rispettivamente.</li>
	<li>Utilizzare una cromatografia liquida di interazione idrofobica (HILIC)-colonna glycan UPLC a una temperatura di 60 ° C per l'analisi della colonna.</li>
	<li>Preparare il solvente A diluendo 50 mL della soluzione madre (soluzione di formiato di ammonio 1 M, pH 4.5) con 950 mL di liquida cromatografia spettrometria di massa (LCMS)-acqua di grado. Preparare la soluzione di riserva come segue:<ol>
<li>Aggiungere 43 mL di acido formico a 700 mL di LCMS-grade H2O.</li>
		<li>Regolare il pH a 4.5 aggiunta goccia a goccia di soluzione di ammoniaca acquosa (25% w/w).</li>
		<li>Trasferire il solvente per un cilindro graduato e riempire fino a 1000 mL con LCMS-grade H2O. negozio a 4-6 ° C fino a 3 mesi.</li>
	</ol>
</li>
	<li>Utilizzare acetonitrile LCMS-grado come solvente B.</li>
	<li>Separare gli N- glicani con l'eluizione di pendenza seguente:<ol>
<li>Iniziare aggiungendo il 95% di solvente B in solvente A. imposta la velocità di flusso a 0,5 mL/min da 0 a 44,5 min.</li>
		<li>Da 0 a 6 minuti, gradualmente diminuire la proporzione del solvente B con un gradiente lineare di to78%.</li>
		<li>Da 6 a 44,5 minuti, gradualmente diminuire la proporzione del solvente B con una sfumatura lineare al 55,9%.</li>
		<li>Da 44,5 a 46,5 minuti, rapidamente diminuire la proporzione del solvente B con una sfumatura lineare dal 55,9% allo 0% e tenere allo 0% per 2 min e avviare il lavaggio della colonna. Ridurre il tasso di flusso a 0,25 mL/min da 44,5 a 55 min.</li>
		<li>Lavare la colonna ulteriormente aumentando solvente B al 95% da 48,5 a 50,5 minuti e tenere al 95% per 4,5 min.</li>
		<li>Riequilibrare la colonna per le condizioni di partenza del solvente B al 95% a 0,5 mL/min da 50,5 a 57 min.</li>
	</ol>
</li>
	<li>Iniettare 45 µ l del campione nel sistema UPLC.</li>
	<li>Eluire gli N- glicani di UPLC con tempi di ritenzione tra 15 e 40 min.</li>
	<li>Raccogliere ogni frazione di picco UPLC osservati usando provette da centrifuga 2 mL e asciugare i campioni per evaporazione centrifugo.</li>
	<li>Iniettare circa 1 pmol di 2-AB etichettato destrano standard (2−20 unità di glucosio) per calibrare la pianta -N- glycan profilatura e di assegnare loro tempi di eluizione in unità di glucosio standardizzati.</li>
</ol>7. MALDI-TOF MS Analysis
<ol><li>Sciogliere i campioni da passo 6,8 a 5 µ l di LCMS-acqua di grado. Mescolare 1 µ l della soluzione campione e 1 µ l di soluzione (ATT) (0,3% p/v in acetonitrile acquosa 70% v/v) 6-aza-2-thiothymine e pipetta il composto sul porta-campioni MALDI-TOF.</li>
	<li>Analizzare i campioni utilizzando uno strumento di spettrometro di massa MALDI-TOF in modalità ioni positivi premendo il pulsante Start .</li>
	<li>Analizzare gli spettri di massa utilizzando il software di analisi MALDI-TOF-MS accompagnamento.</li>
	<li>Interpretare gli spettri utilizzando un opensource glycan interpretazione software19. Impostare i parametri di ricerca per 2-AB etichettatura, [M + Na]+e impostare il parametro di precisione Da 2.0.</li>
</ol>

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sample+preparation+methodology+for+plant+N-.glycan+analysis+using+acid-stable+PNGase+H+.">Rapid sample preparation methodology for plant N-.glycan analysis using acid-stable PNGase H+.</a> Journal of Agricultural and Food Chemistry. 63 (48), 10550-10555 (2015).</li><li>Wang, T., Hu, X. -. C., Cai, Z. -. 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Il protocollo che abbiamo presentato qui consente il confronto dei profili N- glycan di varie cultivar di ravanello. Un notevole vantaggio di questo metodo rispetto ai protocolli esistenti è che è necessaria alcuna modifica di buffer tra il rilascio enzimatico di N- glicani e la reazione di derivatizzazione con 2-AB. Il punto più critico di questa procedura è la purificazione di N- glicani utilizzando la colonna SPE, come la mancata rimozione di sali o altre impurità nella miscela di reazi...

N- glicani in piante hanno attirato l'attenzione aumentata negli ultimi anni, come la ricerca precedente ha evidenziato N- glicani come una potenziale fonte di reazioni crociate immunologiche che può provocare reazioni allergiche1,2 . È stato dimostrato in precedenza che N- glicani su glicoproteine vegetali può influire sulle attività catalitica3,4, termostabilità e pieghevole5,6 o localizzazione subcellulare e secrezione7. Al fine di correlare le strutture glycan con le rispettive funzioni, N- glicani deve essere rilasciata prima da glicoproteine chimicamente o enzimaticamente. Il classico metodo chimico per il rilascio di N- e O- glicani è β-eliminazione, in cui il trattamento alcalino campione è accompagnata da riduzione con boroidruro di cedere un alditol8. Tuttavia, questa procedura preclude etichettatura con un fluoroforo e causa decadimento significativo di unità monosaccaridiche dall'estremità riducente della struttura glycan. Deglycosylation chimico basato su carbonato di ammonio idrossido/trattamento è anche un metodo alternativo comunemente usato9. Nessuno di questi metodi di rilascio chimico degrada le proteine intatte, che permette l'analisi di spettrometria di massa di adenoide glycan piscine senza l'interferenza di frammenti peptidici nello stesso intervallo di massa. Tuttavia, uno svantaggio di questi metodi è il tasso di degradazione aumentata di α1, 3-fucosylated N- glicani, una struttura comune di carboidrato trovata in piante10. In alternativa, i metodi di rilascio enzimatico utilizzando peptide:N- glycanases (PNGases, CE 3.5.1.52) sono anche ampiamente applicato. Ricombinante PNGasi F (da Flavobacterium meningosepticum) è la scelta più comune e permetta la liberazione di tutti i tipi di N- glicani, tranne le strutture recanti un nucleo α1, fucosio 311,12. Pertanto, PNGasi A (isolato da semi di mandorla) viene solitamente utilizzato per l'analisi della pianta N- glicani13. Tuttavia, questo enzima deglycosylates solo proteoliticamente-derivati glicopeptidi ed è in grado di deglycosylate nativo glicoproteine14. Quindi, un workup di multi-step campione è richiesto prima di ulteriori analisi, che provoca perdita di glicani, specialmente quelli di abbondanza bassa15. L'obiettivo generale del metodo è di presentare un workflow ottimizzato per N- glycan rilascio e fluorescenza etichettatura in modo semplice e robusto. La logica sottostante è che PNGasi H+, che è stato recentemente scoperto in Terriglobus roseus e può essere recombinantly espresso in e. coli, possono idrolizzare N- glicani direttamente dal patibolo della proteina in acidi condizioni16. Un vantaggio chiave di utilizzo rispetto ai metodi alternativi di PNGasi H+ è che le reazioni d'etichettatura fluorescente possono essere eseguite nello stesso tubo di reazione senza modificare il buffer di reazione17,18. Le condizioni di preparazione semplice e alto recupero di basso-abbondanza oligosaccaridi fanno di questo metodo un prezioso strumento nell'analisi di N- glicani. Questo protocollo è adatto per l'analisi di N- glicani da varie specie di piante.

<table><tbody><tr><td>&lt;strong&gt;Chemicals:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>SCR, Shanghai</td><td>80132618</td><td></td></tr><tr><td>Acetic acid glacial</td><td>Huada, Guangzhou</td><td>64-19-7</td><td></td></tr><tr><td>Acetonitrile</td><td>General-reagent</td><td>G80988C</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Energy chemical</td><td>W810031</td><td></td></tr><tr><td>2-aminobenzamide</td><td>Heowns, Tianjin</td><td>A41900</td><td></td></tr><tr><td>Sodium cyanoborohydride</td><td>J&amp;amp;K Scientific Ltd</td><td>314162</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Huada, Guangzhou</td><td>67-68-5</td><td></td></tr><tr><td>2AB-labeled dextran ladder, 200 pmol</td><td>Agilent Technologies</td><td>AT-5190-6998</td><td></td></tr><tr><td>6-Aza-2-thiothymine&amp;nbsp;</td><td>Sigma</td><td>275514</td><td></td></tr><tr><td>&lt;strong&gt;Tools/Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Kitchen blender</td><td>Bear, Guangzhou</td><td>LLJ-A10T1</td><td></td></tr><tr><td>Centrifuge</td><td>Techcomp</td><td>CT15RT</td><td></td></tr><tr><td>Centrifugal Evaporator</td><td>Hualida, Taicang</td><td>LNG-T120</td><td></td></tr><tr><td>SPE column</td><td>Supelco</td><td>Supelclean ENVI Carb SPE column</td><td></td></tr><tr><td>MALDI-TOF mass spectrometer</td><td>Bruker</td><td>Autoflex</td><td></td></tr><tr><td>&lt;strong&gt;HPLC Analysis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>High-recovery HPLC vial</td><td>Agilent Technologies</td><td>&amp;nbsp;# 5188-2788</td><td></td></tr><tr><td>HPLC System</td><td>Shimadzu</td><td>Nexera</td><td></td></tr><tr><td>Fluorescence Detector for HPLC</td><td>Shimadzu</td><td>RF-20Axs&amp;nbsp;</td><td></td></tr><tr><td>Column oven</td><td>Hengxin</td><td>CO-2000</td><td></td></tr><tr><td>HPLC Column</td><td>Waters</td><td>Acquity UPLC BEH glycan column</td><td>2.1 &amp;times; 150 mm, 1.7 &amp;mu;m particle size</td></tr><tr><td>LCMS-grade Water</td><td>Merck Millipore</td><td>#WX00011</td><td></td></tr><tr><td>LCMS-grade Acetonitrile</td><td>Merck Millipore</td><td># 100029</td><td></td></tr><tr><td>Formic acid</td><td>Aladdin</td><td>F112034</td><td></td></tr><tr><td>Ammonia solution</td><td>Aladdin</td><td>A112080</td><td></td></tr></tbody></table>

analysis of n-glycans from raphanus sativus cultivars using pngase h+

Negli ultimi anni, le moiety carboidrati delle piante hanno ricevuto una notevole attenzione, sono una fonte potenziale di cross-reattivi, provocando Allergia risposte immunitarie. Inoltre, strutture di carboidrati anche giocare un ruolo critico nel metabolismo vegetale. Qui, presentiamo un metodo semplice e veloce per la preparazione e l'analisi di N- glicani da diverse cultivar di ravanello (Raphanus sativus) usando un N- glycanase specifici per il rilascio delle strutture di carboidrati vegetali. Per raggiungere questo obiettivo, greggio acido tricloroacetico precipitati di ravanello omogeneati erano trattati con PNGasi H+ed etichettati utilizzando 2-aminobenzamide come un tag fluorescente. Con etichetta N- glycan campioni sono stati successivamente analizzati da altissime prestazioni cromatografia liquida (UPLC) separazione e matrix-assisted laser desorption ionizzazione-tempo di spettrometria di massa (MALDI-TOF) volo per una dettagliata strutturale valutazione e di quantificare il relativo abundancies di ravanello-derivati N- glycan strutture. Questo protocollo può essere utilizzato anche per l'analisi di N- glicani da varie altre specie di piante e può essere utile per ulteriori indagini della funzione e gli effetti di N- glicani sulla salute umana.

La figura 1 Mostra una panoramica schematica del protocollo descritto, compreso l'isolamento delle proteine (glico-) dal ravanello, la preparazione di N- glicani, l'analisi UPLC e l'analisi MALDI-TOF-MS di questi componenti. La figura 2 Mostra rappresentative cromatogrammi UPLC di N- glicani derivatizzata delle cultivar ravanello analizzati. La figura 3 Mostra i risultati ottenuti d...

Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

Analisi di N- glicani da Raphanus sativus cultivar utilizzando PNGasi H+

Descriviamo un metodo semplice e veloce per la preparazione e l'analisi di N- glicani da diverse cultivar di ravanello (Raphanus sativus).

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

analysis-of-n-glycans-from-raphanus-sativus-cultivars-using-pngase-h

Research

JoVE Journal

Biochemistry

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

6.5K Views.  Nanjing Agricultural University. Descriviamo un metodo semplice e veloce per la preparazione e l'analisi di N- glicani da diverse cultivar di ravanello (Raphanus sativus).

Video: Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

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 ...

Ricerca

Biochimica

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

Un quantitativo Glycomics e proteomica combinata strategia Purificazione

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Chimica

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

Chemio-enzimatico di sintesi della N- glicani matrice dello sviluppo e dell'anticorpo HIV profilatura

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

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 ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine ​​in cellule di mammifero

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Biologia

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

<meta charset="utf8"></head><body>Profilazione dei tessuti tumorali del colon-retto mediante glicoproteomica guidata dai glifumetti

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

<meta charset="utf8"></head><body>La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi 

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Ricerca sul cancro

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Identificazione e caratterizzazione di glicosilazione delle proteine ​​con specifiche endo-e exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Analisi di N- glicani da Raphanus sativus cultivar utilizzando PNGasi H⁺

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

Un quantitativo Glycomics e proteomica combinata strategia Purificazione

Chemio-enzimatico di sintesi della N- glicani matrice dello sviluppo e dell'anticorpo HIV profilatura

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

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine in cellule di mammifero

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

Identificazione e caratterizzazione di glicosilazione delle proteine con specifiche endo-e exoglycosidases

Analisi di N- glicani da Raphanus sativus cultivar utilizzando PNGasi H+

Riepilogo

Esplora Altri Video

Capitoli in questo video

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

Un quantitativo Glycomics e proteomica combinata strategia Purificazione

Chemio-enzimatico di sintesi della N- glicani matrice dello sviluppo e dell'anticorpo HIV profilatura

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

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine ​​in cellule di mammifero

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

La glicoproteomica guidata dai glifumetti facilita la profilazione completa del glicoproteoma in microambienti tumorali complessi

Identificazione e caratterizzazione di glicosilazione delle proteine ​​con specifiche endo-e exoglycosidases

Analisi di N- glicani da Raphanus sativus cultivar utilizzando PNGasi H⁺

Pulse-chase Analisi di N-linked catene di zuccheri da glicoproteine in cellule di mammifero

Identificazione e caratterizzazione di glicosilazione delle proteine con specifiche endo-e exoglycosidases