Este trabajo fue apoyado por las Tecnologías de la Clínica Proteómica para el cáncer de la iniciativa, 5U24CA126477-04.

1) Prepare lectina conjugada columnas POROS <ol><li> Se puso una máscara para protegerse contra la inhalación de los poros-AL cuentas durante los pasos de 1,1 a 1,2. Pesar la cantidad deseada de granos POROS (100 mg beads/300 volumen final l) y la transferencia en un tubo eppendorf limpio. </li><li> Lávese las cuentas con la adición de 1 mL de buffer fosfato salino (PBS). Pellet cuentas por centrifugación en una microcentrífuga a máxima velocidad durante 3 minutos. Aspirar el sobrenadante y repetir el lavado. </li><li> Pesar la cantidad necesaria de lectina conjugada (1 - 4 mg / l 200 cuentas) y transferir a un tubo eppendorf limpio. Añadir PBS para formar un 5 a 20 mg / ml de solución. Reserva 25 l de esta solución. </li><li> Transferir la solución de lectina restantes a las cuentas poros. Añadir cianoborohidruro de sodio para una concentración final de 50 mM. Coloque el tubo en una mecedora y reaccionar durante la noche a temperatura ambiente. (Nota:. Cianoborohidruro de sodio es tóxico y debe ser manejado en una campana de extracción de residuos contaminados deben eliminarse de manera apropiada.) </li><li> Se precipitan los poros cuentas como en el paso 1.2. Aspirar el sobrenadante y guardar como la solución después de la conjugación. </li><li> Lávese las cuentas con un Buffer enfriamiento ml (1 M Tris-Cl, pH 7,4). Cuentas de pellets como en el paso 1.2 y de manera segura eliminar el sobrenadante. </li><li> Bloquear los sitios restantes reactiva en las cuentas de poros con un búfer de enfriamiento ml. Añadir cianoborohidruro de sodio para una concentración final de 50 mM. Coloque el tubo en una mecedora y se incuban a temperatura ambiente durante 30 min. </li><li> Pellet cuentas como en el paso 1.2 y desechar el sobrenadante. </li><li> Lávese las cuentas con 1 ml de NaCl 1 M. Pellet cuentas y eliminar el sobrenadante. Repetir cuatro veces para un total de cinco lavados. Las cuentas ya están listos para empacar. </li><li> Para determinar la cantidad de proteína que se conjugó con las cuentas de Poros, realizar un ensayo de la concentración de proteínas en las soluciones de la lectina de pre-y post-conjugado conjugados. La diferencia en las concentraciones es la cantidad de proteína que se conjugó con las cuentas. La cantidad de proteína conjugada por volumen de cuentas es igual a la concentración de lectina en los granos. Las concentraciones objetivo lectina son entre 2 -20 mg / ml. </li></ol> 2) Embalaje de la cuentas de lectina conjugada con poros en una columna de PEEK <ol><li> Montar el sistema de empaque (descripción a continuación) y el apoyo en un soporte de anillo de metal. El sistema de embalaje se compone de, de abajo hacia arriba, limitador de presión, columna extremo del acoplador 1, frita, la columna (2 x 50 mm), acoplamiento final la columna 2, conector de columna, columna extremo del acoplador 3, columna (4,5 x 50 mm), columna extremo del acoplador 4 y montaje final. La columna superior sirve como depósito para el material de embalaje. </li><li> Volver a suspender las cuentas de poros en el volumen deseado de un Buffer. (10 mM Tris, pH 7,4, 150 mM de cloruro de sodio, cloruro de calcio 10 mM, 10 mM de cloruro de magnesio) Si una columna de embalaje (~ 200 l de volumen de lecho), resuspender en 400 A. Buffer l </li><li> Transferencia conjugado cuentas POROS en la columna superior (el depósito). Añadir un Buffer para el depósito hasta que el buffer llega a la parte superior de la columna. Coloque con cuidado el conector de extremo en la parte superior de la columna, tratando de evitar burbujas de aire en la columna. Conecte el montaje final de la columna superior a la del sistema HPLC. </li><li> Paquete de la columna por la que fluye el tampón A través del sistema. Comience con un caudal de 0,5 mL / min. Aumentar la tasa de flujo de 0,5 mL / min cada minuto hasta que 4 ml / min o la presión máxima (3000 psi) se ha alcanzado. Continuar en la tasa de flujo máximo posible hasta por lo menos 35 ml de tampón A han pasado por la columna. </li><li> Apague la bomba y deje que la presión en la columna para bajar a &lt;20 psi. </li><li> Suavemente, desmontar el sistema de empaque, a partir de la parte superior. Al final de la columna 2 del acoplador se alcanza, quite cuidadosamente. Parte del material de envasado deberá extrusión de la parte superior de la columna. Con una navaja o filo similar, limpie suavemente lejos de la extrusión de cuentas, dejando una superficie de cuentas que es con la parte superior de la columna. No presione a las perlas mientras hace esto. </li><li> Desenganche la columna de relleno de la parada de anillo. Coloque una frita de nuevo en un acoplador de nuevo final y gire a la columna de relleno más que conectarse con el acoplador de frita / final en el extremo de la columna abierta (anteriormente parte superior). </li><li> Etiqueta de la columna apropiada. Ahora está listo para su uso. Cuando no esté en uso, la columna puede ser almacenada en PBS con azida de sodio 0,02% a 4 ° C durante un máximo de 6 meses. </li></ol> 3) Programa de la HPLC método <ol><li> Los detalles de la programación de la HPLC pueden variar de acuerdo a las especificaciones de software del fabricante. Utilizamos un paradigma Michrom MG4 HPLC. En este equipo, los métodos de crear y acceder en la sección &quot;Configuración&quot; en la parte superior de la pantalla. Programa de la siguiente forma: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tiempo </td><td> Caudal (l / min) </td><td> % A </td><td> % B </td></tr><tr><td> 0:00 </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> Tampón A: 10 mM Tris, pH 7,4, 150 mM de cloruro de sodio, cloruro de calcio 10 mM, 10 mM de cloruro de magnesio Tampón B: 0,5 M de ácido acético El detector de UV debe ser programado para leer a 280 nm a partir de las 0:00 a las 19:50. El eje debe ser ajustado para detectar bajos niveles de absorción, por ejemplo, 0 - 50. UA. </li><li> Si es un muestreador automático de programas disponibles, el siguiente calendario para la recogida de fracción. De lo contrario, recoger las fracciones con la mano como lo indica la hora de realizar la cromatografía. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Fracción </td><td> Demora a la recogida de inyección de la muestra </td><td> Duración de la colección (min) </td></tr><tr><td> Flujo a través de </td><td> 2.8 </td><td> 4.1 </td></tr><tr><td> Obligado </td><td> 9 </td><td> 2.6 </td></tr></tbody></table></li></ol> 4) Preparación de muestras para HPLC <ol><li> Antes de su uso en este protocolo, el plasma humano deberá ser agotado de las 14 proteínas más abundante con una columna de MARS (Agilent, Santa Clara, CA). Agotado plasma, la lactoferrina humana y la invertasa de levadura debe ser individual tripsina digerido y como se describió previamente desalado 1. </li><li> Para preparar la lectina específica de la lactoferrina estándar, realice una cromatografía en 50 ug tripsina digerido lactoferrina sobre cualquiera de la AAL, o la columna SNA utilizando el método en la parte 3. La fracción unida se contienen lectina específica de glicopéptidos lactoferrina. Neutralizar las muestras como en la parte 4.4, y desalar como en la Parte 6. Resuspender la muestra en un ml de tampón A. La muestra resultante está listo para su uso. </li><li> Tres réplicas experimentales se llevarán a cabo. Para preparar la muestra para tres repeticiones, añadir 3 pmol de tripsina digerido invertasa y 1 l de lectina específica de la lactoferrina (ya sea AAL-LAC o SNA-LAC) y 30 l MARS-agotado, tripsina digerido equivalentes de plasma, (PE). PE se define como el volumen de plasma non-processed/intact/original del que se deriva una cantidad dada de procesado de plasma (MARS-agotado y tripsina digerido). Añadir un Buffer de la muestra para llegar a un volumen final de 330 microlitros. </li><li> Añadir tampón de neutralización (1 M Tris pH 8,0), a los viales de muestra que recogerá eluido. El número y el volumen de los viales que trabajan con dependerá de las limitaciones de su cargador de muestras. Para el paradigma MG4, un vial recoge el flujo continuo y dos recoger el eluido. Se requieren aproximadamente 1,5 ml de tampón Tris para neutralizar 1 ml de eluido. PH neutralizado objetivo es 7.0-8.0; prueba del pH prueba final con papel indicador de pH. </li></ol> 5) Realizar la cromatografía <ol><li> Todas las carreras en blanco y la muestra que siguen se utilizará el método descrito en la Parte 1. La columna y tampones están a temperatura ambiente durante su uso. Las columnas se almacenan a 4 ° C, mientras que los amortiguadores son almacenados a temperatura ambiente. </li><li> Sujetar el AAL o la columna de lectina SNA en el sistema HPLC y ejecutar dos métodos en blanco (inyección de búfer sólo una). Asegúrese de que la columna de lectina corresponde a la lactoferrina lectina específica (Parte 2). </li><li> Cromatógrafo de las tres muestras de plasma, la inyección de un espacio en blanco entre cada muestra inyectada, para un total de 7 carreras por HPLC. Las fracciones de carreras en blanco no será preciso recoger. </li></ol> 6) Desalar fracciones recogidas Para cada fracción de utilizar una de 1 ml Agua Oasis HLB SPE cartucho de la siguiente manera: <ol><li> Coloque el número de cartuchos para colector de vacío. </li><li> Húmedo cada cartucho con un ACN mL 80% en 1% de ácido fórmico (medidor de vacío en el colector debe leer 5-20 LNHG). </li><li> Equilibrar los cartuchos con 1,5 ml de ácido fórmico al 0,1% (medidor de vacío en el colector debe leer 5-20 LNHG) </li><li> Volumen de carga total de una muestra en un cartucho (medidor de vacío en el colector debe leer 2 - 2.5. LNHG, y el caudal no debe exceder de 1 ml / min) </li><li> Lavado de los cartuchos con 3 x 1 ml de ácido fórmico al 0,1% (medidor de vacío en el colector debe leer 5-20 LNHG) </li><li> Péptidos eluir / glucopéptidos en tubos eppendorf marcados con una x 1% de acetonitrilo 80 ml en el 0,1% de ácido fórmico. (Medidor de vacío en el colector debe leer 2-2,5 LNHG, y el caudal no debe exceder de 1 mL / min.) Un tubo de recolección solo se debe utilizar para cada cartucho. </li><li> Neutralizar eluido mediante la adición de 60 l de bicarbonato de amonio 0,5 M a cada tubo de recolección. Objetivo neutrpH industrializados se está 7,0-8,0; pH prueba final con papel indicador de pH. </li><li> Concentrado péptidos eluidos / glicopéptidos a ~ 50 l por su ejecución en una centrífuga de vacío (por ejemplo, un Thermo Savant SpeedVac) de ~ 2 horas a 35 ° C. </li></ol> 7) PNGase F-digestión <ol><li> Prueba del pH de la muestra para asegurarse de que es entre 7,0 a 8,0. Si es necesario, añada bicarbonato amónico 50 mM para aumentar el pH. Si el volumen final de la muestra es&gt; 100 l de la muestra, speedvac hasta que esté entre 50 a 100 microlitros. </li><li> Añadir 0,5 l (250 U) de glicerol libre F PNGase a cada tubo de muestra y se incuba a 37 ° C durante la noche. </li></ol> 8) Ejemplos de Código Postal Consejo <ol><li> A partir de PNGase F-digerido muestras, ZipTip cada uno de ellos de la siguiente manera. </li><li> ZipTip húmedo con 10 l de acetonitrilo 100%, seguido de 10 l de acetonitrilo 80%, 0,1% de ácido fórmico. </li><li> Equilibre ZipTip con 20 l de ácido fórmico al 0,1%. </li><li> Cargar la muestra en la ZipTip pipeteando arriba y abajo a través de la matriz ZipTip 10 veces. </li><li> Lave la ZipTip pipeteando 10 l de ácido fórmico al 0,1%, cinco veces. </li><li> Eluir la muestra en un tubo eppendorf limpio con 10 l de acetonitrilo 80%, 0,1% de ácido fórmico. </li><li> Repetir la elución y añadir el segundo 10 l en el mismo tubo. </li><li> Speedvac las muestras a un volumen de &lt;2 l. </li><li> Resuspender en 20 muestras de ácido fórmico l 0,1%. Las muestras ya están listos para ser analizados por LC-MS/MS. </li></ol> 9) Los resultados representativos: La conjugación POROS produce normalmente en concentraciones de lectina de cuentas en el rango de 2 a 20 mg / ml. Esto es óptimo para la cromatografía de afinidad. Cromatografía de lectina de la tripsina digerido plasma MARS-agotado humanos normalmente enriquece glucopéptidos en la fracción unida. Como los glicopéptidos están PNGase F-tratada antes de LC-MS/MS, son identificadas como péptidos con la secuencia consenso de N-ligado, NXS / T (donde X es cualquier residuo, pero prolina), en el que se ha convertido en la asparagina un ácido aspártico por la acción de F. PNGase péptidos con estas características se considera que los péptidos deglicosilada (o &quot;deglycopeptides&quot;). En promedio, el flujo a través de (FT) de la fracción de AAL o cromatografía SNA contiene deglycopeptides 4.2%, mientras que el 30-50% de los péptidos se recuperó en la fracción unida se deglycopeptides. Por lo general, utilizando un Elite QStar, 1000-1300 péptidos se identificaron en la fracción de FT, y 200-400 péptidos se identificarán en la fracción unida. Dos o más deglycopeptides invertasa distintos deben ser observados en la fracción de FT y dos o más distintos deglycopeptides lactoferrina en la fracción unida (Tabla 1). <img src="/files/ftp_upload/1398/1398table.jpg" alt="Tabla 1" /> Tabla 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 proporciona un método rápido para el aislamiento y la identificación de los glucopéptidos en forma de glicanos específicos. Como la glicosilación varía ampliamente dentro de un organismo, en particular durante el desarrollo y patogénesis, esta técnica puede aplicarse para hacer frente a una amplia variedad de preguntas. En particular, estamos usando el método para enriquecer selectivamente glicopéptidos que han sido modificados con cáncer asociado a epítopos como un primer paso hacia el descubrimiento de biomarcadores.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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

Los glicanos son una clase importante de modificaciones post-traduccionales. Por lo general se encuentran en las moléculas secretadas y extracelular, las estructuras de glicanos de la señal del estatuto interno de la célula. Glicanos en las células tumorales tienden a tener el ácido siálico y abundantes restos de fucosa. Proponemos que estas variantes asociadas con el cáncer de glicanos ser explotado para el desarrollo de biomarcadores destinados a diagnosticar enfermedad en estadio inicial. En consecuencia, hemos desarrollado una espectrometría de masas basado en flujo de trabajo que incorpora la cromatografía de afinidad sobre matrices formado a partir de las lectinas, proteínas que se unen las estructuras específicas de glicanos. Las lectinas Sambucus nigra (SNA) y aurantia Aleuria (AAL), que se unen el ácido siálico y fucosa, respectivamente, se acoplaron de forma covalente a cuentas POROS (Applied Biosystems) y se introduce en columnas PEEK para cromatografía líquida de alta presión (HPLC). En pocas palabras, el plasma se agotó de los catorce proteínas más abundantes con un sistema de eliminación de afinidad múltiples (MARS-14; Agilent). Agotado el plasma fue digerido con tripsina y se separaron en el filtrado y fracciones obligado por SNA o HPLC AAL. Las fracciones fueron tratados con PNGaseF para eliminar la N-glicanos ligados, y se analizaron por LC-MS/MS en Elite QStar. Los datos fueron analizados utilizando el software mascota. El diseño experimental incluye controles positivos-fucosilados y sialylated humanos glicopéptidos y lactoferrina negativos los controles de altura glicopéptidos manosa de Saccharomyces cerevisiae, que se utiliza para controlar la especificidad de la captura de lectina. Las principales características de este flujo de trabajo incluyen la reproducibilidad derivado del formato de HPLC, la identificación positiva de los glicopéptidos capturado y PNGaseF tratos de sus motivos Asn-Xxx-Ser/Thr desamidado y evaluación de la calidad con las normas glicoproteína. Optimización del protocolo también incluye la determinación de la proporción adecuada de material de partida a la capacidad de la columna, la identificación de la captura más eficiente y tampones de elución, y el control de la PNGaseF-tratamiento para asegurarse de deglicosilación completo. Direcciones futuras incluyen el uso de este flujo de trabajo para llevar a cabo la espectrometría de masas basado en experimentos de detección en plasma de pacientes con cáncer de mama y el control de los individuos.

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

Lectina conjugada con perlas de Poros fueron empleados para HPLC. Normas glicopéptido sirvieron como controles positivos y negativos. MARS-14 empobrecido, tripsina digerido plasma humano y se sometió a cromatografía de flujo continuo (FT) y las fracciones recogidas obligado para el análisis de ESI-LC-MS/MS. Glicopéptidos fueron enriquecidos en la fracción unida en comparación con el FT.

Una lectina Método HPLC para enriquecer péptidos selectivamente glicosilada de muestras biológicas complejas

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

a-lectin-hplc-method-to-enrich-selectively-glycosylated-peptides-from

Research

JoVE Journal

Biology

15.9K vistas.  University of California, San Francisco - UCSF. Lectina conjugada con perlas de Poros fueron empleados para HPLC. Normas glicopéptido sirvieron como controles positivos y negativos. MARS-14 empobrecido, tripsina digerido plasma humano y se sometió a cromatografía de flujo continuo (FT) y las fracciones recogidas obligado para el análisis de ESI-LC-MS/MS. Glicopéptidos fueron enriquecidos en la fracción unida en comparación con el FT.

Video: 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.

De la onda T de movilidad de iones-espectrometría de masas: Basic Procedimientos experimentales para el análisis de proteínas complejas

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. ...

Investigación

Biología

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.

Péptidos de la Biblioteca de Phage Display modular la expresión génica en las células mesenquimales y potenciar la osteogénesis en defectos óseos unicorticales

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).

Una basada en PCR genotipificación método para distinguir entre las variedades de tipo silvestre y 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.

La lógica, la Plaza experimentales, y el potencial de la Biosíntesis heteróloga Producto Natural Con la eritromicina Complejo antibiótico producido a través de un<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.

Un método para la microinyección de<em&gt; Patiria minata</em&gt; Los cigotos

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.

Purificar el Impuro: Secuenciación metagenomes y Metatranscriptomes de complejos animales asociados a las muestras

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.

Un híbrido método de extracción de ADN para la Evaluación de las comunidades bacterianas cualitativa y cuantitativa de Aves de Corral en muestras de producción

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.

La eliminación de materiales exógenos de la parte exterior de congelados Núcleos para Investigar la antigua comunidades biológicas albergaba el interior

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.

Preparación y observación de muestras biológicas mediante el escaneo de grueso Electrónica de Transmisión de Positrones

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 el etiquetado de las transcripciones en los Escherichia coli las células para una sola molécula de la fluorescencia In Situ hibridación experimentos