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>

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Os autores não têm nada para divulgar.

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 reação podem afetar negativamente a eficiência de derivatização de fluorescência. O método apresentado aqui apenas permite a avaliação da abundância relativa de diferentes N-os glicanos; quantificação absoluta requereria a adição de um padrão interno (como maltopentose) no passo 2.1.
Alternativamente, o agente de derivatização utilizado aqui (2-AB) pode ser facilmente modificado usando 2-aminopiridina (2-AP) ou outros agentes de derivatização. Os obtidos N- glicano derivados da etapa 5.3 podem também ser separados por cromatografia de exclusão (SEC), forte ou fraco ânion troca cromatografia (SAX ou cera), ou métodos HPLC de fase reversa (embora provavelmente com resolução inferior). Para além disso, verifique se as estruturas do N- os glicanos, tratamentos exoglycosidase sequencial podem ser usados após etapa 5.3 para caracterizar a estrutura das N- glicanos com mais detalhes. Finalmente, as amostras da etapa 6,8 poderiam ser analisadas mais usando MALDI-TOF-MSn-com base em métodos de fragmentação. Possíveis aplicações futuras do nosso protocolo incluem a investigar os efeitos de saúde e função de N- os glicanos de rabanete ou outras espécies de plantas.

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.

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	<tbody>
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			<td height="21" style="height:21px;width:347px;">Chemicals:</td>
			<td style="width:123px;"></td>
			<td style="width:207px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid&#160;</td>
			<td>SCR, Shanghai</td>
			<td>80132618</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid glacial</td>
			<td>Huada, Guangzhou</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>General-reagent</td>
			<td>G80988C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid</td>
			<td>Energy chemical</td>
			<td>W810031</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-aminobenzamide</td>
			<td>Heowns, Tianjin</td>
			<td>A41900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cyanoborohydride</td>
			<td>J&#38;K Scientific Ltd</td>
			<td>314162</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide</td>
			<td>Huada, Guangzhou</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2AB-labeled dextran ladder, 200 pmol</td>
			<td>Agilent Technologies</td>
			<td>AT-5190-6998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Aza-2-thiothymine&#160;</td>
			<td>Sigma</td>
			<td>275514</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kitchen blender</td>
			<td>Bear, Guangzhou</td>
			<td>LLJ-A10T1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Techcomp</td>
			<td>CT15RT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifugal Evaporator</td>
			<td>Hualida, Taicang</td>
			<td>LNG-T120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SPE column</td>
			<td>Supelco</td>
			<td>Supelclean ENVI Carb SPE column</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>Autoflex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>&#160;# 5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Column oven</td>
			<td>Hengxin</td>
			<td>CO-2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Waters</td>
			<td>Acquity UPLC BEH glycan column</td>
			<td>2.1 &#215; 150 mm, 1.7 &#956;m particle size</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Water</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td># 100029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Aladdin</td>
			<td>F112034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 estruturas 2AB-derivatizado N- glicano usando espectrometria de massa MALDI-TOF. A Figura 4 mostra a composição quantitativa de N- os glicanos de cada cultivar de rabanete.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57979/57979fig1.jpg"> Figura 1 : Visão esquemática do método análise descrito para os N- glicanos lançados de glicoproteínas rabanete. O método inclui a preparação de proteínas (Glico-) e os N- glicanos, UPLC perfis e análise de MALDI-TOF-MS. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57979/57979fig2.jpg"> Figura 2 : N -glicano perfis de 2-AB rotulado N - os glicanos. Os N- glicanos são isolados do rabanete vermelho, verde rabanete, nabo gegeol, daikon, rabanete cereja e rabanete melancia. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> 
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57979/57979fig3.jpg"> Figura 3 : Análise espectrométrica de massa N - glicano amostras isoladas por uma-UPLC. Os N- glicanos em cada espectrograma são coletados pelo pico do mesmo. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig3large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a> 
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57979/57979fig4.jpg"> Figura 4 : Composição quantitativa de N - os glicanos de cada cultivar de rabanete. Os tamanhos dos círculos e crescentes representam a abundância relativa as 2AB-rotulado N- os glicanos de glicoproteínas de rabanete. <a href="https://www.jove.com/files/ftp_upload/57979/57979fig4large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

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

6.5K 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+

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Projeto Experimental de Laser Microdissecção RNA-Seq: Lições de uma análise do desenvolvimento foliar do milho

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Bioquímica

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

Análise de uma única partícula de alta resolução a partir de imagens de crio-microscopia de elétrons usando SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Isolamento de Proteínas de Filamento Intermediário a partir de Tecidos Múltiplos de Rato para Estudar Modificações Pós-Translacionais associadas ao Envelhecimento

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular choroid layer. Different regions of the retina are anatomically and molecularly distinct, facilitating unique functions and demonstrating differential susceptibility to disease. Proteomic analysis of each of these regions and layers can provide vital insights into the molecular process of many diseases, including Age-Related Macular Degeneration (AMD), diabetes mellitus, and glaucoma. However, separation of retinal regions and layers is essential before quantitative proteomic analysis can be accomplished. Here, we describe a method for dissection and collection of the foveal, macular, and peripheral retinal regions and underlying RPE-choroid complex, involving regional punch biopsies and manual removal of tissue layers from a human eye.One-dimensional SDS-PAGE as well as downstream proteomic analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), can be used to identify proteins in each dissected retinal layer, revealing molecular biomarkers for retinal disease.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

Dissecação da Retina humana e RPE coroide para análise proteômica

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

Medição do consumo de oxigênio mitocondrial nas fibras Permeabilized da drosófila usando quantidades mínimas de tecido

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Dinâmica proteómica e miRNA análise de polissomos de coração de rato isolado após Langendorff perfusão

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

Análise de fucosilado humanos leite trissacarídeos biotecnológicas contexto usando geneticamente codificado biosensores

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

Estudo sobre o metabolismo de seis inseticidas sistêmicas em uma cultura de suspensão celular recém-estabelecida derivada do chá (Camellia sinensis L.) Folhas

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Isolamento de Histone do tecido da folha de sorgo para perfil de espectrometria de massa de top down de potenciais marcadores epigenéticos

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

Preparação de filmes de suporte à amostra em microscopia eletrônica de transmissão usando um bloco de flutuação de suporte

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...