We thank SP Gygi and GM Church for help and guidance. This work was supported by a CNRS chaire d'excellence to ACT.

NOTA: Este método foi descrito anteriormente 12. 1. Isolamento de Proteínas Prepare 1 mg de proteína celular por lise das células, de preferência, por meio de métodos físicos, tais como a prensa francesa, grânulo batimento, ou de ultra-sons. Evitar a lise celular mediada por lisozima porque a enzima irá confundir medições de espectrometria de massa. 2. Precipitação de TCA de Proteínas Adicionar 1 volume de ácido tricloroacético (TCA) a proteína de 4 volumes e frio em gelo durante 10 min para precipitar as proteínas. Centrifugar a 12.000 xg durante 5 min a 4 ° C e remove-se o sobrenadante. Ressuspender o sedimento em 1 ml de acetona gelada e centrifugar a 12000 xg durante 5 min a 4 ° C. Remover o sobrenadante e inverter o tubo no banco para secar o sedimento durante 15 minutos. Peletes de proteína Armazenar a -80 ° C. 3. Desnaturar proteínas e Reduzir dissulfeto Bonds Resuspender as proteínas para ~ 2 mg / ml em 500 ul de tampão de desnaturação e de redução (ou ureia 4 M ou 3% de SDS em 50 mM de HEPES pH 8,5, 5 mM de DTT). Opcionalmente, incluem um inibidor da protease no buffer. Incubar as proteínas durante 30 minutos a 56 ° C, seguido de 10 min à temperatura ambiente. 4. Aklylate grupos livres Sulfidrila para interromper irreversivelmente Formação Dissulfeto de Bond Prepare fresco 0,3 M iodoacetamide na água. ATENÇÃO! Iodoacetamida é altamente tóxico. Adicionar 25 ul de 0,3 M iodoacetamida (15 mM de concentração final) a 500 ul de proteína e incubar durante 20 min no escuro à temperatura ambiente. Extingue-se por adição de iodoacetamida 10 ul de 300 mM DTT (5 mM de concentração final de DTT). Armazenar proteínas alquiladas a -80 ° C. 5. Digestão de proteínas TCA precipitar as proteínas (tal como descrito na etapa 2) e ressuspender em 1 ml de HEPES 50 mM (pH 8,2), 1 M de ureia. Prepare uma solução estoque de Lisil endoproteinase (Lys-C) em água a uma concentração de 2 mg / mL e adicionam-se 5 ul da solução de proteína. Incubar a mistura durante 16 horas à temperatura ambiente. Assegurar a concentração final Lys-C é de 10 ng / mL e a relação de proteína para LysC (w / w) é de 1/50 a 1/200. Ressuspender 20 ug de tripsina de grau de sequenciação em 40 ul de 50 mM de ácido acético, adicionam-se 5 uL (10 ug de tripsina) a Lis-C digest, e incubar durante 6 horas a 37 ° C. Usar a mesma concentração de protease e os rácios de protease-proteína para Lis-C, tal como utilizado para a tripsina. 6. De fase inversa Peptide Extracção <ol><li> Acidifica-se péptidos por adição de ácido trifluoroacético (TFA) até uma concentração final de 0,5% (pH ≈ 2). Anexar uma coluna C18 de um colector de extração. Utilize a maior taxa de fluxo possível para todos os passos excepto a carga e eluição dos peptídeos. </li><li> Wet coluna com 6 ml de acetonitrila (ACN). Lavar coluna com 6 ml de 80% de ACN, 0,1% de TFA, em seguida equilibrar-se com 6 ml de TFA a 0,1%. Não permita que ocoluna para secar entre as etapas. </li><li> Pare a pressão de vácuo e carregar 500 ug de péptidos para a coluna a uma taxa de fluxo de aproximadamente 1 ml / min. Uma vez que os péptidos se ligaram à coluna, a reinicialização vácuo e lavar com 6 mL de TFA a 0,1%, em seguida com 3 ml de tampão de ácido cítrico (ácido cítrico 0,09 M, 0,23 M de Na 2 HPO 4, pH 5,5). Nota: os valores mais elevados de péptidos podem ser marcados, mas a coluna C18 capacidade de ligação deve ser pelo menos duas vezes maior do que a quantidade de péptido para evitar a perda de amostra. </li></ol> 7. On-coluna Peptide Labeling por redutora dimetilação (Redi Labeling) Execute esta etapa sob um capuz químico como cianeto de hidrogênio é liberado em baixa concentração durante o processo de rotulagem. <ol><li> Prepare 12 ml de &quot;leves&quot; e &quot;pesados&quot; buffers REDI de metilato aminas peptídeo livres. Tampão Redi Luz consiste de 0,8% de formaldeído e 0,12 M cianoborohidreto de sódio levando hidrogênios em poeir distribuições isotópicas naturais em tampão de ácido cítrico. Tampão Redi pesada consiste em 0,8% de formaldeído deuterado e 0,12 M deuterado cianoborohidreto de sódio em tampão de ácido cítrico. </li><li> Incubar coluna contendo peptídeos por adição de 10 ml ou de luz ou de tampão Redi pesada para as colunas contendo péptidos em taxa de fluxo de 1 ml / min e repetir a assegurar a rotulagem completa. Lavar a coluna com 6 ml de TFA a 0,1% e, em seguida, com ácido acético a 1 ml de 0,5%. </li><li> Pare vácuo e eluir péptidos marcados em primeiro lugar com 1 mL de 40% de ACN, 0,5% de ácido acético, em seguida com 1 mL de 80% de ACN, 0,5% de ácido acético, utilizando uma taxa de fluxo de aproximadamente 0,5 ml / min. Se desejar, medir a eficiência de marcação de amostras individuais por espectrometria de massa antes de misturar amostras leves e pesados ​​(ver &quot;Os resultados representativos&quot;). Misturar 01:01 amostras de péptidos pesados ​​e etiquetados-luz para ser quantificada por espectrometria de massa. </li></ol> 8. Mistura Peptide separado por pH básico invertida Fase cromatografia (BPRP) &lt;/ P&gt; PH básico de fase reversa (BPRP) cromatografia para separar a mistura de péptidos em várias fracções, que são analisadas de forma independente por LC-MS/MS para aumentar a cobertura do proteoma. <ol><li> Fraccionar a mistura de péptidos numa coluna C18 de HPLC através da aplicação de um gradiente de concentração crescente de ACN em 10 mM de bicarbonato de amónio (pH 8). Comece com 5% (v / v) de ACN durante 5 minutos, para aumentar a 35% de ACN em 60 min, e em seguida a 90% de ACN em 1 min. Reter a ACN 90% durante 4 minutos antes de reduzir o ACN a 5% para re-equilibrar a coluna durante 9 min. Recolha 96 fracções de igual volume de uma placa de 96 poços (de A1 a H12). Monitorar a fraccionamento utilizando um detector de UV a 220 nm, enquanto que os péptidos são eluição da coluna (10-70 min para as condições aqui descritas). </li><li> Combine frações de poços A1, C1, E1 e G1 (fração A1), a partir de poços B1, D1, F1 e H1 (fração B1), a partir de poços A2, C2, E2 e G2 (fração A2) e, consequentemente, para a demais frações. Remover o solvent usando uma centrífuga de vácuo. Ressuspender a partir de fracções de péptidos de A1, B2, A3, B4, A5, B6, A7, B8, A9, B10, A11, B12 e em 130 ul de 1 M urea/0.5% TFA e purificar usando StageTips como descrito no passo 9. Loja frações B1, A2, B3, A4, B5, A6, B7, A8, B9, A10, B11, A12 e à temperatura de -20 º C. </li></ol> 9. Purify Peptídeos por parar e ir Extraction (StageTips) Prepare C18-7 StageTip microcolunas embalando 200 dicas mL pipeta com duas C18 discos com um diâmetro interno (ID) de 1,07 mm. Coloque Dicas Stage em tubos Eppendorf. Use uma microcentrífuga para lavar dicas com 130 mL de metanol, em seguida, 130 ul de 80% ACN, ácido acético 0,5%. Equilibrar StageTips com 130 mL TFA 0,1%. Transferir a mistura de péptidos de StageTips e lava-se com 130 ul de 0,1% de TFA, em seguida, 40 ul de 0,1% de TFA, em seguida, 40 ul de ácido acético a 0,5%. Eluir péptidos primeiro com 20 ul de 40% ACN, 0,5% de ácido acético, em seguida, 20 ul de 80% ACN, 0,5% de ácido acético. Combine um eluatosnd seco por filtração sob vácuo. 10. Microcapilares LC-MS/MS <ol><li> Dissolve-se os péptidos em 1-5 mL de ácido fórmico a 5%, 5% de ACN a uma concentração de aproximadamente 1 mg / mL. Resolver ~ 1 ug péptidos sobre uma 100 um × 20 cm, C18-invertidos coluna de HPLC de fase com um gradiente de 6-22% de ACN em ácido fórmico a 0,125% aplicados sobre 75 ou 100 min a uma taxa de fluxo de ~ 300 nl / min. </li><li> Identificar peptídeos usando um LTQ Orbitrap Velos 12 ou plataforma semelhante cromatografia líquida-espectrometria de massa com um espectrômetro de massa fornecendo alta resolução e precisão de massa de alta. Operar o espectrômetro de massa no modo dependente de dados com uma completa varredura MS (resolução de 60.000) adquirido no analisador Orbitrap. Gerar ion trap linear espectros MS / MS para as 20 mais abundantes iões detectados no espectro de MS completa. Definir controle de ganho automático (AGC) metas para 1 x 10 6 para o pleno MS e 2.000 para MS / MS. Definir máximas vezes acúmulo de íons de 1.000 mspara MS e 150 ms para MS / MS. Excluir íons peptídeo precursor fragmentadas de uma maior seleção de MS / MS para 20-60 seg. </li></ol> 11. MS / MS de Aquisição de Dados Identificar peptídeos comparando espectros MS / MS arquivos RAW para um banco de dados teóricos com um algoritmo como SEQUEST 8 usando esses parâmetros (Tabela 1). Tabela 1. Peptídeo de banco de dados da pesquisa Parâmetros. <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> Parâmetros Gerais </td><td> digestão totalmente tryptic com até 2 perdeu clivagens </td></tr><tr height="19"><td> Tolerância íon precursor ppm 25 </td></tr><tr height="19"><td> 1.0 tolerância íon fragmento Da </td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> Modificações estáticos </strong&gt; </td><td> Da 57,02146 em cisteína, carboxyamidomethylation </td></tr><tr height="19"><td> Da 28,03130 em lisina e o péptido N-terminal, a etiqueta dimetilação luz </td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> Modificações dinâmicas </td><td> Da 15,99491 em metionina, a oxidação </td></tr><tr height="19"><td> 6,03766 Da em lisina e o péptido N-terminal, a etiqueta dimetilação pesado </td></tr></tbody></table><ol><li> Filtrar péptidos a uma taxa de detecção falso 1% com um método tal como a estratégia de 9-alvo chamariz utilizando uma base de dados de estruturas de leitura abertas nas orientações efectivos e invertidas. </li></ol> 12. Peptide Quantificação Calcular as áreas de pares pesados ​​e leves de MS1 extraído íon cromatogramas (áreas de pico MS1) e peptídeo sinal-ruído (S / N) proporções 10. Incluir pares de peptídeos somente quando sua média de sinal-noiproporção se for superior a cinco. Quantificar a abundância relativa de um peptídeo das duas amostras como a razão entre as áreas dos picos de MS1 versões pesadas e leves do mesmo péptido (razão de área de pico MS1). Calcular as abundâncias relativas de proteína como a razão da área do pico MS1 mediana para todos os péptidos da proteína.

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Os autores declaram não competindo interesses financeiros ou outros conflitos de interesse.

Vários pontos tornar a rotulagem de isótopos estáveis ​​de peptídeos usando um método atraente para proteômica quantitativa dimetilação redutora (rotulagem Redi): reagentes baratos de rotulagem (reagentes custam menos de US $ 1 por amostra), a taxa de reação rápida (~ 10 min), ausência de produtos secundários, altas reprodutibilidade (Figuras 3, 4), produtos de reacção estáveis, capacidade de usar qualquer protease, e alta eficiência de ionização de peptídeos identificados. Marca...

Medindo diferenças de concentração de muitas proteínas entre as amostras complexas é um desafio central em proteômica. Cada vez mais, isso é feito através da marcação de proteínas de cada uma das amostras com diferentes marcações isotópicas, combinando as amostras, e por espectrometria de massa para quantificar as diferenças de concentração. Existem vários métodos para a marcação isotópica estável de proteínas e peptídeos. 15N rotulagem 1 e 2 SILAC introduzir marcadores isotópicos metabolicamente in vivo, ao passo que iCAT 3, iTRAQ 4, e redução dimetilação 5 adicionar tags de isótopos estáveis ​​após a extracção de proteína e de digestão. Entre esses métodos, dimetilação redutora (rotulagem Redi) está ganhando popularidade como um método barato e reprodutível para quantificar as diferenças de concentração de proteínas em quase qualquer tipo de amostra. Rotulagem Redi envolve péptidos que reagem com o formaldeído, para formar uma base de Schiff, a qual é então reduzidapor cianoborohidreto. Esta reacção dimethylates grupos amino livres no terminal N e cadeias laterais de lisina e monomethylates prolinas N-terminais. O protocolo aqui descrito metila péptidos em amostra 1 com um rótulo de &quot;luz&quot; usando reagentes com átomos de hidrogénio na sua distribuição isotópica natural e a amostra 2, com um rótulo &quot;pesada&quot; usando formaldeído deuterado e cianoborohidreto (Figura 1). Cada grupo amino dimetilado sobre um péptido resulta numa diferença de massa de 6,0377 Da entre luz e formas, pesados, que são utilizados para distinguir entre as duas formas, utilizando um espectrómetro de massa. Especificamente, as abundâncias relativas de péptidos são quantificadas como a razão entre as áreas de cromatogramas de iões MS1 extraídos (MS1 razão de área de pico) da luz e da versão pesada para cada par de iões de péptidos. A abundância relativa de uma proteína é calculada como a razão da área do pico MS1 média entre todos os péptidos da proteína. Neste relatório, nós descrevemos um protocolo robusto para condutaing Redi experiências de marcação por LC-MS/MS, que inclui o péptido de extracção de fase reversa em fase sólida, marcação Redi na coluna, o péptido de fraccionamento por pH básico de fase reversa (BPRP) cromatografia, e purificação de misturas de péptidos utilizando StageTips (Figura 2) . Discutimos vantagens e limitações do uso de rotulagem Redi para proteômica quantitativa.

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		<tr height="19">
			<td height="19" style="height:19px;width:377px;">trichloroacetic acid</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:136px;">T9159</td>
			<td style="width:319px;">protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetone</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">650501</td>
			<td>protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71736</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">S8045</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">43816</td>
			<td>denature, reduce, alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">protease Inhibitor Complete Mini Cocktail</td>
			<td>Roche</td>
			<td>4693124001</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I6125</td>
			<td>alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">H7523</td>
			<td>resuspend, extract, label protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">C5670</td>
			<td>resuspend protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">Lysyl Endoprotease</td>
			<td>Wako Chemicals</td>
			<td style="width:136px;">129-02541</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sequencing grade trypsin</td>
			<td>Promega</td>
			<td style="width:136px;">V5111</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">320099</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">tC18 Sep-Pak C18 cartridges</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">extraction manifold</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">14261</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cyanoborohydride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71435</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">deuterated formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>492620</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium cyanoborodeuteride</td>
			<td>CDN isotopes</td>
			<td style="width:136px;">D-1797</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18-HPLC column (4.6 x 250 mm, 5 &#181;m particle size)</td>
			<td>Agilent</td>
			<td style="width:136px;">770450-902</td>
			<td>basic pH reversed-phase chromatography</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">399388</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18 Empore Disks</td>
			<td>3M</td>
			<td style="width:136px;">14-386-3&#160;</td>
			<td>STAGE tips</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">methanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">494437</td>
			<td>STAGE tips</td>
		</tr>
	</tbody>
</table>

quantitative proteomics using reductive dimethylation for stable isotope labeling

Rotulagem isótopo estável de péptidos por dimetilação redutiva (marcação Redi) é um método para quantificar com precisão as diferenças entre as amostras de expressão de proteínas utilizando espectrometria de massa. Rotulagem Redi é realizada usando regular (luz) ou deuterados formas (pesados) de formaldeído e sódio cianoborohidreto para adicionar dois grupos metil para cada amina livre. Aqui demonstramos um protocolo robusto para a rotulagem Redi e comparação quantitativa de misturas complexas de proteínas. As amostras de proteínas para comparação são digeridas em péptidos, rotuladas para transportar a luz ou etiquetas de metilo pesados, misturados, e co-analisados ​​por LC-MS/MS. Abundância relativa de proteína são quantificados por comparação de áreas de pico do cromatograma de iões de versões marcadas pesadas e leves do péptido constituinte extraído da MS espectro completo. O método descrito aqui inclui a preparação de amostras por extração em fase sólida de fase reversa, Redi rotulagem na coluna de peptídeos, peptídeos fracionamento por rev básico pHfase ersed (BPRP) cromatografia, e StageTip purificação peptídica. Discutimos vantagens e limitações de rotulagem Redi em relação a outros métodos de incorporação de isótopos estáveis. Destacamos novas aplicações usando rotulagem Redi como um método rápido, barato e preciso para comparar a abundância de proteínas em quase qualquer tipo de amostra.

Avaliou-se a exatidão, precisão e reprodutibilidade de rotulagem Redi usando Saccharomyces cerevisiae e Clostridium phytofermentans lisados ​​de células inteiras. Primeiro, quantificou o Redi eficiência de marcação de uma mistura de C. phytofermentans lisados ​​de proteína a partir de celulose (pesado marcado, H) e glicose (rótulo de luz, L) culturas. Quando filtrada para uma taxa de falsa descoberta peptídeo 1%, esta amostra continha 11.194 sequências de peptídeos exclusivo...

Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Rotulagem de isótopos estáveis ​​de peptídeos por dimetilação redutora (rotulagem Redi) é uma estratégia rápida e barata para proteômica precisos baseados em espectrometria de massa quantitativos. Aqui demonstramos um método robusto para a preparação e análise de misturas de proteínas, utilizando a abordagem de Redi que pode ser aplicado a quase todos os tipos de amostra.

Proteômica quantitativa utilizando redutora dimetilação para Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

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Research

JoVE Journal

Chemistry

16.0K visualizações.  Genoscope.  Rotulagem de isótopos estáveis ​​de peptídeos por dimetilação redutora (rotulagem Redi) é uma estratégia rápida e barata para proteômica precisos baseados em espectrometria de massa quantitativos. Aqui demonstramos um método robusto para a preparação e análise de misturas de proteínas, utilizando a abordagem de Redi que pode ser aplicado a quase todos os tipos de amostra. 

Vídeo: Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Grande Escala Profiling não-alvo metabolômica de soro por cromatografia líquida de ultra-espectrometria de massa (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Química

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

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

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

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

Melhorou em-gel redutivas β-eliminação para Comprehensive O-ligados e sulfo-glycomics por Espectrometria de Massa

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

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

O exame quantitativo e qualitativo dos Interações partícula-partícula coloidal Usando Probe Nanoscopia

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

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 ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Normas para Análise Quantitativa Metalloproteomic Usando Tamanho Exclusão ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Peptídeos e proteínas quantificação usando Automated imuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Em grande escala proteômica de cima para baixo usando zona capilar eletroforese espectrometria de massa em Tandem

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

Multiplexação automatizada da amostra usando rotulagem isotópica precursora combinada e marcação isobárica (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

Suspensões aquosas estáveis de aglomerados de ferrite de manganês com dimensão e composição de nanoescala tunable

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

Identificação de órgãos primários em plantas medicinais usando marcação de 13C6-glicose

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

Autor em destaque: Perfil de órgãos primários de plantas usando marcação de 13C6-glicose e LC-MS

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

13C6-Marcação de glicose associada a LC-MS: Identificação de órgãos primários de plantas na síntese de metabólitos secundários

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...