Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
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Este trabajo fue apoyado en parte por la Fundación de Ciencias naturales de China (la concesión número 31471703, A0201300537 y 31671854 a J.V. y L.L., número 31470435 de la concesión a G.Y.) y el Plan talentos extranjeros 100 (número de licencia JSB2014012 a J.V.).

1. muestras
<ol><li>Adquirir diferentes variedades de fresco rábano (Raphanus sativus L.).</li>
</ol>2. aislamiento de proteína de rábano
<ol><li>Homogeneizar aproximadamente 100 g de rábanos frescos con una licuadora de cocina durante 10 minutos.</li>
	<li>Transferir la mezcla a un tubo de centrífuga de 50 mL y centrifugar a 14.000 × g a 4 ° C por 20 min eliminar el material insoluble.</li>
	<li>Transferir el sobrenadante con cuidado en un nuevo tubo de centrífuga de 50 mL y añadir un volumen igual de solución de ácido tricloroacético (TCA) de 2 M. Nota: Agregar TCA precipita soluble (glico) proteínas.</li>
	<li>Centrifugar a 14.000 × g a 4 ° C por 30 min y retire el sobrenadante de las concentrados (glico) proteínas.</li>
	<li>Lavar el precipitado con 20 mL de agua desionizada y centrifugar a 14.000 × g a 4 ° C por 5 min eliminar polisacáridos y oligosacáridos solubles.</li>
	<li>Repita el paso 2.5. cuatro veces.</li>
	<li>Vuelva a suspender el pellet en 1 mL de agua desionizada y transfiéralo a un tubo de centrífuga 1.5ml fresco.</li>
</ol>3. preparación de N- glicanos
<ol><li>Mezcle 50 μl de solución de proteína de paso 2.7. (equivalente a 5 g de rábanos frescos) con 0,2 mU de recombinante PNGasa H+ en 10 mM de ácido acético.</li>
	<li>Incubar la mezcla de reacción a 37 ° C por 12 h. Después de la incubación, centrifugar a 14.000 × g por 5 minutos quitar la proteína adicional y enzima.</li>
	<li>Transferir el sobrenadante a un tubo de centrífuga 1.5ml fresco.</li>
</ol>4. purificación de N- glicanos
<ol><li>Preparar la columna de extracción en fase sólida (SPE) para enriquecer los N- glicanos y quitar sales y otras impurezas de la mezcla de reacción, aumentando la selectividad del fluorógenos 2-Aminobenzamida (2-AB) etiquetado agente de N- glicanos derivatización.<ol>
<li>Añadir 3 mL de agua desionizada para lavar la columna.</li>
		<li>Añadir 3 mL de acetonitrilo 80% solución de ácido trifluoroacético (TFA, 0.1% v/v) para activar la columna SPE.</li>
		<li>Añadir 3 mL de agua desionizada para equilibrar la columna SPE.</li>
	</ol>
</li>
	<li>Transferir la muestra en el paso 3.3 a la columna y descartar el flujo a través.</li>
	<li>Añadir 1,5 mL de agua desionizada para lavar la columna y descartar el flujo a través.</li>
	<li>Eluir los N- glicanos liberados de la columna con 1,5 mL de solución acuosa de acetonitrilo 20%, que contiene 0.1% TFA (v/v) en un tubo de centrífuga de 2 mL.</li>
	<li>Quitar el solvente por evaporación centrífugo a temperatura ambiente. Evaporar hasta que la muestra esté completamente seca.</li>
</ol>5. fluorescencia derivatización de N- glicanos
<ol><li>Preparar 1 mL de solución 2-AB, que consta de 35 mM 2-AB y 0.1 M cianoborohidruro de sodio en solución de ácido acético/sulfóxido de dimetil (7:3, v/v).</li>
	<li>Añadir 5 μl de solución 2-AB para las muestras secadas (paso 4.5.) deriva de seis diferentes rábanos. Vortex cada muestra hasta que totalmente disuelto.</li>
	<li>Incubar la mezcla por 2 h a 65 ° C.</li>
	<li>Deje enfriar el muestra a temperatura ambiente durante 5 minutos y añadir 5 μl de agua desionizada y 40 μl de acetonitrilo. Centrifugar los tubos a 14.000 x g durante 3 minutos.</li>
	<li>Transferir 48 μl del sobrenadante en un frasco HPLC de alta recuperación μl 300. Almacenar las derivatizados N- glycan muestras a-20 ° C hasta por un mes.</li>
</ol>6. HILIC UPLC perfiles de N- glicanos
<ol><li>Analizar las muestras usando un sistema UPLC estándar conectado a un detectorset de fluorescencia en línea en las longitudes de onda de excitación/emisión de 330 nm/420 nm, respectivamente.</li>
	<li>Utiliza una cromatografía de líquidos de interacción hidrofóbica (HILIC)-columna de glicanos UPLC a una temperatura de columna de 60 ° C para el análisis.</li>
	<li>Preparar A solvente diluyendo 50 mL de solución madre (solución de formiato de amonio de 1 M, pH 4,5) con 950 mL de líquido cromatografía-espectrometría de masas (LCMS)-grado de agua. Prepare la solución como sigue:<ol>
<li>Añadir 43 mL de ácido fórmico a 700 mL de LCMS-grado H2O.</li>
		<li>Ajustar el pH a 4.5 por adición gota a gota de solución de amoníaco acuosa (25% w/w).</li>
		<li>Transferir el solvente a una probeta y llenar hasta 1000 mL con LCMS-grado H2O. almacenar a 4-6 ° C hasta por 3 meses.</li>
	</ol>
</li>
	<li>Utilizar acetonitrilo grado LCMS como solvente B.</li>
	<li>N- glycans se separan con la siguiente elución gradiente:<ol>
<li>Comience agregando el 95% de disolvente B en solvente A. Ajuste la velocidad de flujo de 0,5 mL/min de 0 min 44,5.</li>
		<li>Del minuto 0 al minuto 6, disminuyen progresivamente la proporción del disolvente B % to78 gradiente lineal.</li>
		<li>De 6 min a min 44,5, disminuyen progresivamente la proporción del disolvente B con un degradado lineal a 55,9%.</li>
		<li>De min min 46,5 44,5, rápidamente disminuir la proporción de solvente B con un degradado lineal de 55,9% a 0% y mantener en 0% por 2 min e iniciar lavado de la columna. Reducir la tasa de flujo de 0.25 mL/min de 44,5 a 55 min.</li>
		<li>Lavar la columna adicional incrementando el disolvente B a 95% de min min 50,5 48,5 y sostenga en el 95% de 4.5 min.</li>
		<li>Volver a equilibrar la columna para las condiciones iniciales de 95% de disolvente B en 0.5 mL/min de 50.5 a min 57.</li>
	</ol>
</li>
	<li>Inyectar al sistema UPLC 45 μl de la muestra.</li>
	<li>Eluir los N- glicanos por UPLC con tiempos de retención entre 15 y 40 minutos.</li>
	<li>Recoger cada fracción de pico UPLC observado usando tubos de centrífuga de 2 mL y secar las muestras por evaporación centrífugo.</li>
	<li>Inyectar aproximadamente 1 pmol de etiquetado estándar de dextrano (2−20 unidades de glucosa) para calibrar la plantaN- glycan perfiles y asignar los tiempos de elución en unidades de glucosa estandarizada 2-AB.</li>
</ol>7. uso de MALDI-TOF MS análisis
<ol><li>Disuelva las muestras de paso 6.8 en 5 μl de LCMS-agua de grado. Mezclar 1 μl de la solución de muestra y 1 μl de la solución de (ATT) (0,3% w/v en acetonitrilo acuoso 70% v/v) 6-aza-2-thiothymine y pipeta de la mezcla en el portamuestra de MALDI-TOF.</li>
	<li>Analizar las muestras usando un instrumento de espectrómetro de masas MALDI-TOF en ion positivo modo presionando el botón Start .</li>
	<li>Analizar los espectros de masas mediante el software de análisis de MALDI-TOF-MS que lo acompaña.</li>
	<li>Interpretar los espectros utilizando un código abierto glycan interpretación software19. Definir los parámetros de búsqueda para 2-AB etiquetado [M + Na]+y establecer el parámetro de exactitud Da 2.0.</li>
</ol>

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El protocolo que hemos presentado aquí permite la comparación de los N- glicanos perfiles de distintas variedades de rábano. Una importante ventaja de este método en comparación con los protocolos existentes es que no hay cambios de tampón entre la liberación enzimática de N- glicanos y la reacción de derivatización con 2 AB son necesarios. El paso más crítico de este procedimiento es la purificación de N- glycans utilizando la columna SPE, como fracaso para eliminar las sales o im...

N- glycans en plantas han atraído la atención creciente en los últimos años, como la investigación anterior ha puesto de manifiesto N- glicanos como una fuente potencial de reacciones cruzadas inmunológicas que pueden provocar las respuestas alérgicas1,2 . Se ha demostrado previamente que N- glycans en glicoproteínas vegetales pueden afectar actividad catalítica3,4, termoestabilidad y plegable de5,6 o localización subcelular y secreción7. Con el fin de correlacionar las estructuras glicanos con sus respectivas funciones, N- glycans debe primero liberarse de glicoproteínas enzimático o químicamente. El método químico clásico para la liberación de N- y O- glicanos es β-eliminación, en la cual tratamiento alcalino de la muestra se acompaña de reducción con hidruro de boro para producir un alditol8. Sin embargo, este procedimiento impide etiquetar con un fluoróforo y causa decaimiento significativo de unidades de monosacárido del extremo reductor de la estructura de glicanos. Deglycosylation química basada en el tratamiento de carbonato de hidróxido de amonio es también un método alternativo utilizado9. Ninguno de estos métodos de liberación químico degrada proteínas intactas, que permite el análisis de espectrometría de masa de glicanos piscinas sin la interferencia de los fragmentos de péptido en el mismo rango de masa. Sin embargo, una desventaja de estos métodos es la tasa de degradación creciente de α1, 3 fucosylated N- glycans, una estructura común de carbohidratos encontrados en las plantas10. Como alternativa, métodos de liberación enzimática utilizando péptidos:N- glicanasas (PNGases, EC 3.5.1.52) se aplican también extensamente. Recombinante PNGasa F (de Flavobacterium meningosepticum) es la opción más común y permite la liberación de todos los tipos de N- glicanos, excepto las estructuras teniendo un núcleo α1, fucosa 311,12. Por lo tanto, A PNGasa (aislada de semillas de almendra) se utiliza generalmente para el análisis de planta N- glycans13. Sin embargo, esta enzima deglycosylates sólo proteolytically derivados de glicopéptidos y es incapaz de deglycosylate nativo glicoproteínas14. Por lo tanto, es necesaria un workup muestra varios pasos antes de análisis, que causa pérdida extensa de glycans, especialmente aquellos de baja abundancia15. El objetivo del método es presentar un flujo de trabajo optimizado para el lanzamiento de N- glicanos y fluorescencia de etiquetado de una manera sencilla y robusta. La lógica subyacente es que PNGasa H+, que fue descubierto recientemente en Terriglobus roseus y puede ser por vía recombinante expresada en e. coli, puede hidrolizar N- glycans directamente desde el andamio de proteínas en ácidos condiciones16. Una ventaja clave de usar PNGasa H+ sobre métodos alternativos es que reacciones Etiquetadoras fluorescentes se pueden realizar en el mismo tubo de reacción sin modificar la reacción buffers17,18. Las condiciones de preparación simple y alta recuperación de baja abundancia oligosacáridos hacen este método una valiosa herramienta en el análisis de N- glicanos. Este protocolo es adecuado para el análisis de N- glicanos de varias especies de plantas.

<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+

En los últimos años, las moléculas de hidratos de carbono de las plantas han recibido considerable atención, ya que son una fuente potencial de respuesta inmune cruz-reactivo, que provoca la alergia. Además, estructuras de hidratos de carbono también juegan un papel fundamental en el metabolismo vegetal. Aquí, presentamos un método simple y rápido para la preparación y análisis de N- glicanos de diferentes variedades de rábano (Raphanus sativus) con un N- glycanase específico para la liberación de las estructuras de carbohidratos de origen vegetal. Para lograr esto, los precipitados de homogenados de rábano crudo ácido tricloroacético fueron tratados con PNGasa H+y etiquetados utilizando 2-Aminobenzamida como una etiqueta fluorescente. Las etiqueta N- glycan muestras posteriormente fueron analizadas por ultra rendimiento cromatografía líquida (UPLC) separación e ionización-tiempo de desorción láser asistida por matriz de la espectrometría de masas de vuelo (MALDI-TOF) para un detallado estructural evaluación y cuantificar abundancies relativos de las estructuras derivadas de rábano N- glicanos. Este protocolo también puede utilizarse para el análisis de N- glicanos de varias otras especies de plantas y puede ser útil para la investigación adicional de la función y los efectos de N- glicanos en la salud humana.

La figura 1 muestra una descripción esquemática del protocolo descrito, incluyendo el aislamiento de (glico) proteínas de rábano, la preparación de N- glicanos, el análisis UPLC y el MALDI-TOF-MS en análisis de estos componentes. La figura 2 muestra representativos cromatogramas UPLC de N- glycans derivatizados de los cultivares analizados rábano. La figura 3 muestra los res...

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+

Análisis de N- glicanos de Raphanus sativus variedades usando PNGasa H+

Describimos un método simple y rápido para la preparación y análisis de N- glicanos de diferentes variedades de rábano (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+

6.6K vistas.  Nanjing Agricultural University. Describimos un método simple y rápido para la preparación y análisis de N- glicanos de diferentes variedades de rábano (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.

Caracterización estructural de polisacáridos de pared celular mananos en plantas con ritmo

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

Investigación

Bioquímica

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 Glicómica cuantitativa y Proteómica estrategia de purificación combinada

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

Química

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.

Chemo-enzimático síntesis de N- glicanos para el desarrollo de la matriz y el anticuerpo del VIH perfiles

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.

Pulso-caza de análisis de cadenas de azúcares unidos a N de glicoproteínas en células de mamífero

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

Biología

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>Perfil de tejidos de cáncer colorrectal mediante glicoproteómica guiada por glicómica

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 glicoproteómica guiada por glucómica facilita el perfil completo del glicoproteoma en microambientes tumorales complejos

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 glicoproteómica guiada por glucómica facilita el perfil completo del glicoproteoma en microambientes tumorales complejos 

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

Investigación sobre el cáncer

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.

Identificación y caracterización de la glicosilación de proteínas específicas de uso de endo-y exoglicosidasas

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

Análisis de N- glicanos de Raphanus sativus variedades usando PNGasa H⁺

Resumen

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Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

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