Este trabalho foi financiado pela Clinical Technologies Proteômica para o cancro da iniciativa, 5U24CA126477-04.

1) Prepare colunas lectina-conjugada POROS <ol><li> Colocar uma máscara para se proteger contra a inalação de POROS-AL contas durante as etapas de 1,1-1,2. Pesar a quantidade desejada de contas POROS (100 mg mL de volume beads/300 final) e transferir para um tubo eppendorf limpo. </li><li> Lavar contas com adição de 1 mL tampão fosfato (PBS). Contas de pelotas por centrifugação em microcentrífuga a velocidade máxima por 3 minutos. Remover o sobrenadante e repetir a lavagem. </li><li> Pesar a quantidade desejada de lectina não conjugada (1-4 mg / 200 mL contas) e transferir para um tubo eppendorf limpo. Adicionar PBS para formar uma 5-20 mg / mL. Reserve 25 mL desta solução. </li><li> Transferir a solução lectina restantes para as contas POROS. Adicionar cyanoborohydride de sódio a uma concentração final de 50 mM. Colocar o tubo em um rocker e reagir durante a noite a temperatura ambiente. (Nota:. Cyanoborohydride de sódio é tóxico e deve ser tratado em um exaustor resíduos contaminados devem ser eliminados de forma adequada.) </li><li> Pellet as contas POROS como no passo 1.2. Remover o sobrenadante e salvar como a solução de pós-conjugação. </li><li> Lavar contas com um buffer Quenching mL (1 M Tris-Cl, pH 7,4). Contas da pelota como no passo 1.2 e segurança descartar o sobrenadante. </li><li> Bloquear os sites restantes reativa nas contas POROS com um buffer Quenching mL. Adicionar cyanoborohydride de sódio a uma concentração final de 50 mM. Colocar o tubo em um rocker e incubar em temperatura ambiente por 30 min. </li><li> Contas da pelota como no passo 1.2 e descartar o sobrenadante. </li><li> Lavar contas com 1 mL 1 M NaCl. Contas de pelotas e descartar o sobrenadante. Repita quatro vezes para um total de cinco lavagens. As contas estão agora prontos para embalar. </li><li> Para determinar a quantidade de proteína que foi conjugado com as contas POROS, realizar um ensaio de concentração de proteínas nas soluções de pré-lectina conjugada e pós-conjugados. A diferença nas concentrações é a quantidade de proteína que foi conjugado com as contas. A quantidade de proteína conjugada por volume de talão é igual a concentração de lectina nas esferas. Concentrações lectina-alvo são entre 2 -20 mg / mL. </li></ol> 2) as contas de embalagem lectina-conjugada POROS em uma coluna PEEK <ol><li> Montar o sistema de embalagem (descrição abaixo) e apoiá-lo em um suporte de anel de metal. O sistema de embalagem consiste em, de baixo para cima, restritor de pressão, coluna final acoplador coluna 1, fritas, (2 x 50 mm), acoplador final coluna 2, conector de coluna, coluna final acoplador 3, coluna (4,5 x 50 mm), final 4 acoplador coluna e montagem final. A coluna superior serve como um reservatório para o material de embalagem. </li><li> Volte a suspender as contas POROS no volume desejado de tampão A. (10 mM Tris, pH 7,4, 150 mM cloreto de sódio, cloreto de cálcio 10 mM, 10 mM de cloreto de magnésio) Se uma coluna de embalagem (~ 200 mL volume do leito), ressuspender em 400 mL de buffer A. </li><li> Transferência de contas conjugado POROS na coluna superior (reservatório). Adicionar tampão A para o reservatório até que o buffer atinge o topo da coluna. Delicadamente, coloque a montagem final para o topo da coluna, tentando evitar as bolhas de ar na coluna. Ligar a montagem final da coluna superior para o sistema de HPLC. </li><li> Encher a coluna pelo fluxo de buffer A através do sistema. Comece com um fluxo de 0,5 mL / min. Aumentar a vazão de 0,5 mL / min a cada minuto até que 4 mL / min ou a pressão máxima (3000 psi) foi atingido. Continue com o caudal máximo possível até, pelo menos, 35 mL de tampão A passaram pela coluna. </li><li> Desligar as bombas e permitir que a pressão sobre a coluna cair para &lt;20 psi. </li><li> Delicadamente, desmontar o sistema de embalagem, a partir do topo. Quando o fim 2 acoplador coluna é atingido, retire com cuidado. Algum material deve ser embalado de extrusão a partir do topo da coluna. Com uma lâmina de barbear ou borda afiada similar, gentilmente limpe as contas de extrusão, deixando uma superfície das esferas que é mesmo com a parte superior da coluna. Não aplique pressão para as contas ao fazer isto. </li><li> Desengatar a coluna empacotada do suporte do anel. Coloque uma nova fritas em um acoplador novo final e vire a coluna empacotada mais para conectar este com o acoplador fritas / fim na extremidade coluna aberta (antiga topo). </li><li> O título da coluna apropriada. Ele agora está pronto para uso. Quando não em uso, a coluna pode ser armazenada em PBS com azida de sódio 0,02% a 4 ° C por até 6 meses. </li></ol> 3) Programa de método HPLC <ol><li> Os detalhes da programação do seu HPLC irá variar de acordo com as especificidades do software do fabricante. Nós usamos um Paradigma Michrom HPLC MG4. Nesta máquina, os métodos são construídas e acesso no âmbito do &quot;Setup&quot; guia na parte superior da tela. Programa o seguinte método: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tempo </td><td> Vazão (mL / min) </td><td> % A </td><td> B% </td></tr><tr><td> 00:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 09:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 09:01 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13:50 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13:51 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr><tr><td> 19:50 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr></tbody></table> Um buffer: 10 mM Tris, pH 7,4, 150 mM cloreto de sódio, cloreto de cálcio 10 mM, 10 mM de cloreto de magnésio Tampão B: 0,5 M de ácido acético O detector de UV deve ser programado para leitura a 280 nm 0:00-19:50. O eixo y deve ser ajustado para detectar baixos níveis de absorção, por exemplo, 0 -. 50 UA. </li><li> Se um amostrador automático é um programa, disponível o seguinte calendário para a coleta de fração. Caso contrário, recolha as frações à mão, como indicado na execução cromatografia. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Fração </td><td> Atraso à recolha de injeção de amostra </td><td> Duração da coleta (min) </td></tr><tr><td> Fluxo através </td><td> 2,8 </td><td> 4,1 </td></tr><tr><td> Obrigado </td><td> 9 </td><td> 2,6 </td></tr></tbody></table></li></ol> 4) Prepare amostras para HPLC <ol><li> Antes de usar neste protocolo, plasma humanos devem ser esgotados os 14 proteínas mais abundantes usando uma coluna MARS (Agilent; Santa Clara, CA). Esgotados plasma humano, lactoferrina e invertase de levedura deve ser individualmente tripsina-digerido e dessalgado conforme descrito anteriormente 1. </li><li> Para preparar a lectina específica lactoferrina padrão, execute cromatografia em 50 ug de tripsina-digerida lactoferrina mais tanto o AAL ou a coluna SNA usando o método na Parte 3. A fração ligada conterá glicopeptídeos lectina específica lactoferrina. Neutralize as amostras como na parte 4.4, e dessalinizar como na Parte 6. Ressuspender a amostra em 1 mL tampão A. A amostra obtida está pronta para uso. </li><li> Três réplicas experimentais será realizada. Para preparar amostra para três repetições, adicionar 3 pmol de tripsina-digerida invertase e 1 ml de lectina específica lactoferrina (ou AAL-LAC ou SNA-LAC) a 30 mL MARS-empobrecido, tripsina-digerida equivalentes, plasma (PE). A PE é definido como o volume de plasma non-processed/intact/original a partir do qual uma determinada quantidade de processados ​​plasma (MARS-empobrecido e tripsina digerido) foi derivada. Adicionar tampão A para a amostra para chegar a um volume final de 330 mL. </li><li> Adicionar tampão de neutralização (1 M Tris pH 8,0), para os frascos de amostra que irá recolher eluído. O número eo volume de frascos de você trabalhar com dependerá das limitações de seu amostrador automático. Para a Paradigm MG4, um frasco de coleta do fluxo através e dois recolher o eluato. Vai exigir cerca de 1,5 mL tampão Tris para neutralizar 1 mL de eluato. PH neutralizado é alvo 7,0-8,0; pH teste teste final com papel indicador de pH. </li></ol> 5) Realizar cromatografia <ol><li> Todas as execuções em branco e da amostra que se seguem vai usar o método descrito na Parte 1. A coluna e os amortecedores estão em temperatura ambiente durante o uso. As colunas são armazenadas a 4 ° C, enquanto que os amortecedores são armazenados em temperatura ambiente. </li><li> Acople a AAL ou a coluna lectina SNA para o sistema de HPLC e executar dois métodos em branco (injeção de tampão de apenas A). Garantir que a coluna lectina corresponde à lactoferrina lectina específica (Parte 2). </li><li> Cromatógrafo a três amostras de plasma, injetando uma em branco entre cada amostra injetado, para um total de 7 é executado HPLC. Frações de corridas em branco não precisam ser recolhidos. </li></ol> 6) dessalinizar coletadas frações Para cada fração de usar um 1 mL Waters Oasis HLB SPE cartucho da seguinte forma: <ol><li> Anexar o número de cartuchos necessários para múltiplas vácuo. </li><li> Wet cada cartucho com 1 mL ACN 80% em 1% de ácido fórmico (vacuômetro em múltiplas deve ler 5-20 lnHg). </li><li> Equilibrar cartuchos com 1,5 mL de ácido fórmico 0,1% (vacuômetro em múltiplas deve ler 5-20 lnHg) </li><li> Volume total de carga de uma amostra em um cartucho (Vacuômetro em múltiplas deve ler 2 -. LnHg 2,5, ea taxa de fluxo não deve exceder 1 mL / min) </li><li> Lavar com 3 cartuchos de 1 mL x ácido fórmico 0,1% (Vacuômetro em múltiplas deve ler 5-20 lnHg) </li><li> Peptídeos eluir / glicopeptídeos em tubos eppendorf rotulados com 1 x 1 mL de acetonitrila 80% em 0,1% de ácido fórmico. (Vacuômetro em múltiplas deve ler 2-2,5 lnHg, ea taxa de fluxo não deve exceder 1 mL / min.) Um tubo de coleta única deve ser usado para cada cartucho. </li><li> Neutralize eluído pela adição de 60 mL 0,5 M de bicarbonato de amónio para cada tubo de coleta. Alvo neutrpH é desmineralizada é 7,0-8,0; pH teste final com papel indicador de pH. </li><li> Concentre-se peptídeos eluídos / glicopeptídeos para ~ 50 mL por executá-los em uma centrífuga a vácuo (por exemplo, a Thermo Savant SpeedVac) por aproximadamente 2 horas @ 35 ° C. </li></ol> 7) PNGase F-digestão <ol><li> PH da amostra de teste para garantir que ele está entre 7,0-8,0. Se necessário, adicione bicarbonato de amônio 50 mM para aumentar o pH. Se o volume da amostra final é&gt; 100 da amostra, mL speedvac até que esteja entre 50-100 mL. </li><li> Adicionar 0,5 mL (250 U) de glicerol livre F PNGase a cada tubo de amostra e incubar a 37 ° C durante a noite. </li></ol> 8) amostras Dica Zip <ol><li> Começando com PNGase F-digerida amostras, ziptip cada um como se segue. </li><li> Ziptip molhado com 10 mL de acetonitrila 100%, seguido por 10 mL de acetonitrila 80%, 0,1% de ácido fórmico. </li><li> Equilibrar ziptip com 20 mL de ácido fórmico 0,1%. </li><li> Carga da amostra para a ziptip pipetando cima e para baixo através da matriz ziptip 10 vezes. </li><li> Lave o ziptip pipetando 10 mL de ácido fórmico 0,1%, 5 vezes. </li><li> Eluir a amostra em um tubo eppendorf limpo com 10 mL de acetonitrila 80%, 0,1% de ácido fórmico. </li><li> Repita a eluição e adicione o mL 10 segundo para o mesmo tubo. </li><li> Speedvac as amostras a um volume de &lt;mL 2. </li><li> Amostras ressuspender em 20 mL de ácido fórmico 0,1%. As amostras estão prontas para análise por LC-MS/MS. </li></ol> 9) Resultados Representante: A conjugação POROS tipicamente rendimentos on-bead lectina concentrações na faixa de 2-20 mg / mL. Isso é ótimo para cromatografia de afinidade. Cromatografia lectina de tripsina-digerida MARS com depleção de plasma humano normalmente enriquece glicopeptídeos na fração ligada. Como a glicopeptídeos são PNGase F-tratados antes de LC-MS/MS, elas são identificadas como peptídeos com a seqüência consenso N-ligados, NXS / T (onde X é qualquer resíduo, mas prolina), em que a asparagina foi convertido para um ácido aspártico através da ação de Peptídeos PNGase F. com essas características são consideradas como peptídeos deglycosylated (ou &quot;deglycopeptides&quot;). Em média o fluxo-through (FT), fração de AAL ou cromatografia SNA contém deglycopeptides 2-4%, enquanto que 30-50% dos peptídeos recuperada na fração ligada são deglycopeptides. Normalmente, usando uma Elite QStar, 1000-1300 peptídeos serão identificados na fração FT, e 200-400 peptídeos serão identificados na fração ligada. Dois ou mais deglycopeptides invertase distintas devem ser observados na fração de FT e dois ou mais deglycopeptides distintas lactoferrina na fração ligada (Tabela 1). <img src="/files/ftp_upload/1398/1398table.jpg" alt="Tabela 1" /> Tabela 1.

<ol>
	<li>Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=multiplexed+assays+for+low+abundance+proteins+in+plasma+by+targeted+mass+spectrometry+and+stable+isotope+dilution.">multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.</a> Mol Cell Proteomics. 6, 2212-2229 (2007).</li></ol>

Este protocolo fornece um método rápido para isolar e identificar glicopeptídeos de forma glicano específico. Como glicosilação varia amplamente dentro de um organismo, particularmente durante o desenvolvimento e patogênese, esta técnica pode ser aplicável para tratar uma grande variedade de perguntas. Em particular, estamos usando o método para seletivamente enriquecer glicopeptídeos que foram modificados com câncer associado epítopos como um primeiro passo para a descoberta de biomarcadores.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Human plasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>We make this in house</td>
</tr>
<tr>
<td>Human lactoferrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L0520</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Yeast invertase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0408</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Oass HLB SPE cartridges</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT094225</td>
<td>1 cc volume</td>
</tr>
<tr>
<td>ZipTip pipet tips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>ZTC1 8M0 96</td>
<td>Millipore, C18 for 10 ml pipette</td>
</tr>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP154-1</td>
<td>99%</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6141</td>
<td>99%</td>
</tr>
<tr>
<td>Calcium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4901</td>
<td>96%</td>
</tr>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>98%</td>
</tr>
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>242853</td>
<td>99.7%</td>
</tr>
<tr>
<td>pH indicator paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M95903</td>
<td>Range 0-14</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A998</td>
<td>99.9%</td>
</tr>
<tr>
<td>Formic acid</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28905</td>
<td>99%</td>
</tr>
<tr>
<td>Phosphate-buffered saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-136</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>71289</td>
<td>99.5%</td>
</tr>
<tr>
<td>PNGase F</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>P0705L</td>
<td>Glycerol-free</td>
</tr>
<tr>
<td>Vacuum-filter flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SCGVU05RE</td>
<td>0.2 mm pores</td>
</tr>
<tr>
<td>POROS-AL beads</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>1-6028-02</td>
<td>&nbsp;</td>
<tr>
<td>Aleuria aurantia lectin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1390</td>
<td>&nbsp;</td>
<tr>
<td>Sambucus nigra agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1300</td>
<td>&nbsp;</td>
<tr>
<td>Sodium cyanoborohydride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>296945</td>
<td>5.0 M solution</td>
</tr>		</tbody>
		</table>
		</div>

a lectin hplc method to enrich selectively-glycosylated peptides from complex biological samples

Glicanos são uma classe importante de modificações pós-translacionais. Tipicamente encontradas em moléculas secretadas e extracelular, as estruturas glicano sinal de estatuto interno da célula. Glicanos em células tumorais tendem a ter ácido siálico abundante e metades fucose. Propomos que essas variantes associadas ao câncer glicano ser explorada para o desenvolvimento de biomarcadores visa diagnosticar doença em estágio inicial. Assim, desenvolvemos uma espectrometria de massa com base em fluxo de trabalho que incorpora cromatografia de afinidade em matrizes formadas a partir de lectinas, proteínas que se ligam glicano estruturas específicas. As lectinas Sambucus nigra (SNA) e Aleuria aurantia (AAL), que se ligam ácido siálico e fucose, respectivamente, foram covalentemente acoplado a contas POROS (Applied Biosystems) e embalados em colunas PEEK para cromatografia líquida de alta pressão (HPLC). Resumidamente, o plasma foi esgotada dos quatorze proteínas mais abundantes usando um sistema de remoção de múltiplos afinidade (MARS-14; Agilent). Esgotados plasma foi tripsina-digerida e separados em flow-through e frações vinculado por SNA ou HPLC AAL. As frações foram tratados com PNGaseF para remover N-glicanos ligados, e analisados ​​por LC-MS/MS em um Elite QStar. Os dados foram analisados ​​usando o software da mascote. O delineamento experimental incluídos controles positivos e-fucosylated sialylated glicopeptídeos e lactoferrina humana negativa controles de alta manose glicopeptídeos de Saccharomyces cerevisiae, que foram utilizados para monitorar a especificidade da captura de lectina. As principais características deste fluxo de trabalho incluem a reprodutibilidade derivado do formato de HPLC, a identificação positiva dos glicopeptídeos capturados e tratados com PNGaseF de seus motivos Asn-Xxx-Ser/Thr desamidados, e avaliação da qualidade usando padrões glicoproteína. Protocolo de otimização também incluiu determinação da relação adequada de matéria-prima para a capacidade da coluna, identificando a captura mais eficiente e buffers de eluição, e monitorar a PNGaseF tratamento para garantir deglycosylation completo. Direções futuras incluem o uso deste fluxo de trabalho para realizar experimentos de espectrometria de massa baseada na descoberta de plasma de pacientes com câncer de mama e indivíduos controle.

Watch this Scientific Journal Video about A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples at JoVE.com

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Contas lectina-conjugada POROS foram empregadas para HPLC. Padrões glicopeptídeo serviram como controle positivo e negativo. MARS-14 empobrecido, tripsina-digerida plasma humano foi cromatografada e flow-through (FT) e frações ligado coletadas para análises ESI-LC-MS/MS. Glicopeptídeos foram enriquecidos na fração ligada quando comparado com FT.

Um método HPLC Lectin enriquecer Peptídeos seletivamente glicosilada de Complexo Amostras Biológicas

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

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Biology

15.9K visualizações.  University of California, San Francisco - UCSF. Contas lectina-conjugada POROS foram empregadas para HPLC. Padrões glicopeptídeo serviram como controle positivo e negativo. MARS-14 empobrecido, tripsina-digerida plasma humano foi cromatografada e flow-through (FT) e frações ligado coletadas para análises ESI-LC-MS/MS. Glicopeptídeos foram enriquecidos na fração ligada quando comparado com FT.

Vídeo: A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T-wave Ion Mobility Spectrometry massa: Basic Procedimentos para Análise Experimental Protein Complex

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Biologia

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Peptídeos de Phage Biblioteca Mostrar modular a expressão de genes em células mesenquimais e potenciar a osteogênese em defeitos ósseos unicorticais

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Um método de genotipagem baseados em PCR para distinguir entre variedades do tipo selvagem e Ornamental de Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

A Lógica, etapas experimentais, e potencial de Biossíntese produto heteróloga Natural Apresentando o complexo antibiótico eritromicina Um produzido através<em&gt; E. coli</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Um Método para microinjeção de<em&gt; Patiria minata</em&gt; zigotos

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

Purificando o Impure: Sequenciação metagenomes e metatranscriptomas do Complexo amostras associadas a animais

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Um método de extração de DNA híbrido para a Avaliação de comunidades bacterianas Qualitativa e Quantitativa de amostras de produção de aves de capoeira

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

Remoção de materiais exógenos da parte exterior dos núcleos congelados para investigar a antiga comunidades biológicas abrigava Dentro

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

Preparação e observação de amostras biológicas Grosso por Scanning Transmission Electron Tomografia

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Método para a rotulagem transcritos em células individuais Escherichia coli para único-molécula fluorescência In Situ as experiências de hibridação