Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
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Este trabalho foi financiado em parte pela Fundação ciência Natural da China (conceder números 31471703, A0201300537 e 31671854, J.V. e ll, conceder número 31470435 para G.Y.) e o plano de talentos estrangeiros 100 (número de concessão JSB2014012 de J.V.).

1. coleta de amostra
<ol><li>Compra de diferentes cultivares de fresco rabanete (Raphanus sativus L.).</li>
</ol>2. isolamento da proteína de rabanete
<ol><li>Homogeneizar a cerca de 100 g de rabanete fresco com um misturador de cozinha para 10 min.</li>
	<li>Transferi o chorume para um tubo de centrífuga de 50 mL e centrifugação a 14.000 × g a 4 ° C por 20 min remover o material insolúvel.</li>
	<li>Transferir o sobrenadante cuidadosamente para um novo tubo de centrifugação de 50 mL e adicionar um volume igual de solução de ácido tricloroacético (TCA) de 2m. Nota: Adicionar TCA irá precipitar proteínas solúveis (glicogénio).</li>
	<li>Centrifugar a 14.000 × g a 4 ° C por 30 min e retirar o sobrenadante as proteínas peletizadas (glicogénio).</li>
	<li>Lave a pelota com 20 mL de água desionizada e centrifugação a 14.000 × g a 4 ° C por 5 min remover polissacarídeos e oligossacarídeos solúveis.</li>
	<li>Repita a etapa 2.5. quatro vezes.</li>
	<li>Re-suspenda as pelotas em 1 mL de água desionizada e transferir para um tubo de centrífuga de 1,5 mL.</li>
</ol>3. preparação da N- os glicanos
<ol><li>Mix 50 µ l da solução da proteína da etapa 2.7. (igual a 5 g de rabanete fresco) com 0,2 mU de recombinantes PNGase H+ no ácido acético de 10 mM.</li>
	<li>Incube a mistura a 37 ° C por 12 h. Após a incubação, centrifugar a 14.000 × g por 5 min remover a proteína extra e a enzima.</li>
	<li>Transferi o sobrenadante para um tubo de centrífuga de 1,5 mL.</li>
</ol>4. purificação de N- os glicanos
<ol><li>Preparar a coluna de extração de fase sólida (SPE) para enriquecer os N- glicanos e remover os sais e outras impurezas da mistura da reação, aumentando a seletividade do fluorogenic 2-aminobenzamide (2-AB) rotulagem agente para N- glicano derivatização.<ol>
<li>Adicione 3 mL de água desionizada para lavar a coluna.</li>
		<li>Adicione 3 mL de solução de acetonitrila 80% contendo ácido trifluoroacético (TFA, 0,1% v/v) para o ativo a SPE-coluna.</li>
		<li>Adicione 3 mL de água desionizada para equilibrar a coluna da SPE.</li>
	</ol>
</li>
	<li>Transferir a amostra da etapa 3.3 na coluna e descartar o escoamento.</li>
	<li>Adicione 1,5 mL de água desionizada para lavar a coluna e descartar o escoamento.</li>
	<li>Eluir os lançado N- os glicanos da coluna usando 1,5 mL de solução aquosa de 20% acetonitrila, contendo 0,1% TFA (v/v) em um tubo de centrífuga de 2 mL.</li>
	<li>Remova o solvente por evaporação centrífuga à temperatura ambiente. Evapore até que a amostra esteja completamente seca.</li>
</ol>5. fluorescência derivatização de N- os glicanos
<ol><li>Prepare-se 1 mL de solução 2-AB, consistindo de 35 mM 2-AB e 0,1 M cyanoborohydride de sódio em solução de ácido acético/sulfóxido de dimetilo (7:3, v/v).</li>
	<li>Adicione 5 µ l de solução 2-AB, para as amostras secas (etapa 4.5.) derivada de seis diferentes rabanetes. Vórtice de cada amostra até completamente dissolvida.</li>
	<li>Incubar a mistura durante 2 h a 65 ° C.</li>
	<li>Deixe a amostra esfriar até à temperatura por 5 min e adicione 5 µ l de água deionizada e 40 µ l de acetonitrilo. Centrifuga os tubos a 14.000 x g, durante 3 min.</li>
	<li>Transferi 48 µ l do sobrenadante para um tubo HPLC de alta recuperação de µ l 300. Armazene as pyrazole N- glicano amostras a-20 º C por até um mês.</li>
</ol>6. UPLC-uma caracterização das N- os glicanos
<ol><li>Analise as amostras usando um sistema UPLC padrão conectado a um detectorset de fluorescência on-line em comprimentos de onda de excitação/emissão de 330 nm/420 nm, respectivamente.</li>
	<li>Use uma cromatografia líquida de interação hidrofóbica (uma)-coluna de glicano UPLC à temperatura de 60 ° C, para a análise de coluna.</li>
	<li>Preparar o solvente A diluir 50 mL da solução (solução de formiato de amônio 1 M, pH 4,5) com 950 mL de líquido cromatografia / espectrometria de massa (LCMS)-grau de água. Prepare a solução-mãe da seguinte maneira:<ol>
<li>Adicionar 43 mL de ácido fórmico a 700 mL de LCMS-classe H2O.</li>
		<li>Ajuste o pH para 4,5 por adição gota a gota de solução aquosa de amoníaco (25% w/w).</li>
		<li>Transferir o solvente para uma proveta graduada e encher a 1000 mL com LCMS-classe H2O. loja a 4-6 ° C por até 3 meses.</li>
	</ol>
</li>
	<li>Usar o LCMS-grau acetonitrilo como solvente B.</li>
	<li>Separe os N- glicanos com a seguinte gradiente eluição:<ol>
<li>Comece adicionando 95% do solvente B em solvente r. conjunto a taxa de fluxo em 0,5 mL/min de 0 para min 44,5.</li>
		<li>De min 0 a 6 min, gradualmente diminua a proporção do solvente B com um % to78 gradiente linear.</li>
		<li>De 6 min a min 44,5, gradualmente diminua a proporção do solvente B com um gradiente linear para 55,9%.</li>
		<li>De 44,5 min a min 46,5, rapidamente diminuir a proporção do solvente B com um gradiente linear de 55,9% para 0% e segurar a 0% por 2 min e iniciar a lavagem da coluna. Reduza a taxa de fluxo para 0,25 mL/min de 44,5 a 55 min.</li>
		<li>Lavar a coluna ainda mais aumentando o solvente B a 95% de 48,5 min a min 50,5 e segurar a 95% para 4,5 min.</li>
		<li>Re-equilibrar a coluna para as condições de partida de 95% solvente B em 0,5 mL/min de 50,5 para 57 min.</li>
	</ol>
</li>
	<li>Injecte 45 µ l da amostra no sistema UPLC.</li>
	<li>Eluir os N- os glicanos por UPLC com tempos de retenção entre 15 e 40 min.</li>
	<li>Recolher a cada fração de pico UPLC observada usando tubos de centrífuga de 2ml e secar as amostras por evaporação centrífuga.</li>
	<li>Injete aproximadamente 1 pmol de 2-AB rotulado dextrano padrão (2−20 unidades de glicose) para calibrar a planta -N- glicano perfis e atribuir seus tempos de eluição em unidades de glicose padronizada.</li>
</ol>7. MALDI-TOF MS Analysis
<ol><li>Dissolver as amostras da etapa 6,8 em 5 µ l de LCMS-grau de água. Misturar 1 µ l da solução da amostra e 1 µ l de solução (ATT) (0,3% p/v em acetonitrila aquosa 70% v/v) 6-aza-2-thiothymine e a pipeta a mistura sobre o transportador de amostra de MALDI-TOF.</li>
	<li>Analise as amostras usando um instrumento de espectrômetro de massa MALDI-TOF em íon positivo modo pressionando o botão Iniciar .</li>
	<li>Analise os espectros de massa usando o software de análise que acompanha MALDI-TOF-MS.</li>
	<li>Interprete os espectros usando uma fonte aberta glicano interpretação software19. Definir os parâmetros de pesquisa para 2-AB etiquetando, [Na + M]+e definir o parâmetro de precisão Da 2.0.</li>
</ol>

<ol>
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Apresentamos aqui o protocolo permite a comparação dos perfis de vários cultivares de rabanete - glicano N. Uma vantagem significativa do método comparado com protocolos existentes é que nenhuma alteração de tampão entre a liberação enzimática de N- os glicanos e a reação de derivatização com 2-AB é necessárias. O passo mais crítico deste procedimento é a purificação do N- os glicanos usando a coluna SPE, como falha para remover os sais ou outras impurezas na mistura de rea...

Os N- glicanos em plantas têm chamado a atenção crescente nos últimos anos, como pesquisas anteriores destacou os N- glicanos como uma fonte potencial de reacções imunológicas que podem provocar respostas alérgicas1,2 . Foi demonstrado anteriormente que os N- glicanos em glicoproteínas de planta pode afetar a atividade catalítica de3,4, estabilidade térmica e dobramento5,6 ou Localização subcellular e secreção7. A fim de correlacionar as estruturas glicano com suas respectivas funções, os N- glicanos deve ser lançados de glicoproteínas ou quimicamente ou enzimaticamente. O método químico clássico para liberar N- e O- os glicanos é β-eliminação, em que o tratamento alcalino amostra é acompanhado por redução com borohidreto para produzir um alditol8. No entanto, este procedimento impede a rotulagem com um fluoróforo e causa deterioração significativa de unidades de monossacarídeo do redutor final da estrutura de glicano. Deglycosylation química, com base no tratamento de hidróxido de amônio/carbonato é também um método alternativo comumente usado9. Nenhum desses métodos de liberação química degrada proteínas intactas, que permite a análise de espectrometria de massa de glicano sem rótulo piscinas sem a interferência de fragmentos de peptídeo na mesma faixa de massa. No entanto, um inconveniente desses métodos é a taxa de aumento da degradação de α1, 3-fucosilado N- os glicanos, uma estrutura comum de carboidrato encontrado em plantas,10. Como alternativa, os métodos de liberação enzimática usando peptídeo:N- glycanases (PNGases, CE 3.5.1.52) são também amplamente aplicados. Recombinante PNGase F (de Flavobacterium meningosepticum) é a escolha mais comum e permite a libertação de todos os tipos de N- os glicanos, exceto as estruturas portadoras de um núcleo α1, fucose 311,12. Portanto, A PNGase (isoladas de sementes amêndoas) geralmente é usada para a análise da planta Nos glicanos -13. No entanto, esta enzima deglycosylates apenas proteoliticamente-derivado glicopeptídeos e é incapaz de deglycosylate nativo glicoproteínas14. Portanto, um exame de amostra de várias etapas é necessário antes de mais análise, que provoca a perda extensa dos glicanos, especialmente aqueles de baixa abundância15. O objetivo geral do método é apresentar um fluxo de trabalho otimizado para N- glicano liberação e rotulagem de forma simples e robusta de fluorescência. A lógica subjacente é que o PNGase H+, que recentemente foi descoberto em Terriglobus roseus e pode ser expressa de recombinantes em e. coli, pode hidrolisar os N- glicanos diretamente do andaime proteína em ácido condições de16. A principal vantagem de usar PNGase H+ sobre métodos alternativos é que reações de rotulagem fluorescentes podem ser executadas em tubo de reação mesmo sem alterar a reação amortecedores17,18. As condições de preparação simples e alta recuperação de oligossacarídeos de baixo-abundância fazem esse método uma ferramenta valiosa na análise do N- os glicanos. Este protocolo é apropriado para a análise de N- os glicanos de várias espécies 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+

Nos últimos anos, as metades de hidrato de carbono de plantas têm recebido atenção considerável, como eles são uma fonte potencial de cross-reactive, alergia, provocando reacções imunológicas. Além disso, estruturas de carboidratos também desempenham um papel fundamental no metabolismo da planta. Aqui, apresentamos um método simples e rápido para preparar e analisar N- os glicanos de diferentes cultivares de rabanete (Raphanus sativus) usando um N- glycanase específico para o lançamento de estruturas de carboidratos de origem vegetal. Para conseguir isso, cru ácido tricloroacético precipitados de rabanete homogenates foram tratados com PNGase H+e rotulados usando 2-aminobenzamide como uma etiqueta fluorescente. N- glicano amostras etiquetadas foram analisadas posteriormente pela separação de ultra desempenho cromatografia líquida (UPLC) e laser assistida por matriz dessorção ionização tempo de voo (MALDI-TOF) espectrometria de massa para uma detalhada estrutural avaliação e a quantificação relativas abundancies das estruturas rabanete-derivados N- glicano. Este protocolo também pode ser usado para a análise de N- os glicanos de várias outras espécies de plantas e pode ser útil para posterior investigação da função e efeitos da N- os glicanos na saúde humana.

A Figura 1 mostra uma visão esquemática do protocolo descrito, incluindo o isolamento de proteínas (Glico-) de rabanete, a preparação de N- os glicanos, a análise UPLC e a análise de MALDI-TOF-MS desses componentes. A Figura 2 mostra representativas cromatogramas UPLC do pyrazole N- os glicanos dos cultivars rabanete analisados. A Figura 3 mostra os resultados obtidos das est...

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+

Análise dos N- glicanos de Raphanus sativus cultivares usando PNGase H+

Nós descrevemos um método simples e rápido para a preparação e análise de N- os glicanos de diferentes cultivares de rabanete (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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

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

6.6K visualizações.  Nanjing Agricultural University. Nós descrevemos um método simples e rápido para a preparação e análise de N- os glicanos de diferentes cultivares de rabanete (Raphanus sativus).

Vídeo: 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.

Caracterização estrutural de polissacarídeos de parede celular Medeiros em plantas usando a ritmo

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

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.

A Quantitative Glycomics e Proteomics Combinada estratégia de purificação

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.

Quimio-enzimático síntese de N- os glicanos para desenvolvimento de matriz e anticorpos de HIV de criação de perfil

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-chase Análise de Cadeias N-linked Açúcar de Glicoproteínas em células de mamíferos

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>Perfil de tecidos de câncer colorretal usando glicoproteômica guiada por glicômicos

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>A glicoproteômica guiada por glicômicos facilita o perfil abrangente do glicoproteoma em microambientes tumorais complexos

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.

A glicoproteômica guiada por glicômicos facilita o perfil abrangente do glicoproteoma em microambientes tumorais complexos 

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

Pesquisa do câncer

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.

Identificação e caracterização de glicosilação de proteínas específicas usando endo-e exoglycosidases

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

Análise dos N- glicanos de Raphanus sativus cultivares usando PNGase H⁺

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