Los autores agradecen el apoyo financiero de las siguientes instituciones: la Fundación Nacional de Ciencias (Grant 0.719.232), el Departamento de Energía (Grant DEFG02-01ER15129), la WM Keck Foundation (subvención 041.904), y la Universidad George Washington, el Fondo de Investigación de mejora. Los autores están agradecidos por las fibras ópticas GeO2 basada generosamente proporcionados por los sistemas de fibra de infrarrojos (Silver Spring, MD), así como para la discusión inicial sobre el Protocolo en relación con el grabado de fibra por Mark E. Reeves y Joan A. Hoffmann, de la George Washington universidad. Los autores también desean agradecer a Jessica A. Stolee por su ayuda durante la grabación en vídeo del protocolo.

1. Los componentes ópticos <ol><li> La ablación con láser de ionización electrospray ablación con láser (LAESI) espectrometría de masas (MS) es producido por un pulso de 5 ns láser de longitud de onda en micras 2,94. Un oscilador óptico paramétrico sintonizable impulsado por un láser Nd: YAG (Opolette 100, Opotek Inc., Carlsbad, CA) se utiliza para la ablación en este estudio. La tasa de repetición se selecciona de entre 5 y 100 Hz. Este es un láser de clase IV, que la exposición directa puede causar graves daños permanentes a los ojos o la piel. La reflexión difusa del haz de láser también puede ser peligroso para la piel o los ojos. Usar un láser gafas adecuadas para su protección. </li><li> La fibra óptica de vidrio basados ​​en dióxido de germanio se obtuvo a partir de infrarrojos Fiber Systems, Inc. (Silver Spring, MD). Si hay Hytrel y revestimientos de poliamida en la fibra siga las siguientes instrucciones para eliminar de los extremos. Calentar 1-metil-2-pirrolidona a 130-150 ° C bajo una campana extractora. Sumerja la fibra de los extremos que se quieren eliminar a este líquido durante aproximadamente 1 minuto o hasta que el recubrimiento comienza a ablandar y despegar. Sumerja la capa blanda en metanol o isopropanol por un minuto, y limpie el recubrimiento sobrante con una toalla sin pelusa. Después de retirar el recubrimiento, unirá los dos extremos de la fibra con una hoja de zafiro (Kitco fibra óptica, Virginia Beach, VA) y al anotar suavemente el ajuste. </li><li> Para lograr la nitidez deseada, químicamente grabar un extremo de la punta de la fibra en un 1% (v / v) de ácido nítrico (grado reactivo) solución. Verticalmente y sumerja el extremo seleccionado de la fibra en la solución de ácido a una profundidad de 300 a 500 m después del contacto inicial. Después de aproximadamente 15 minutos de aguafuerte, la punta de forma espontánea se separará de la superficie del ácido. Esto resulta en una afilada punta de la fibra con radio de ~ 15 m de la curvatura. Enjuague con agua destilada para eliminar cualquier residuo ácido. </li><li> Montaje final grabada de la fibra en un micromanipulador (MN-151, Narishige, Tokio, Japón) y llevarlo a la proximidad (~ 20 m) de la superficie de la célula para la deposición de energía eficiente. </li><li> Sostenga el extremo opuesto al grabado de la fibra óptica por una fibra desnuda tirada (BFC300, Siskiyou Corporation, de Grants Pass, Oregón). Para la alineación óptica, la tirada de fibra se monta en un traductor de cinco ejes (BFT-5, Siskiyou Corporation, de Grants Pass, Oregón). La energía del láser (0,5 a 1 mJ) está acoplado en la fibra de enfocar el haz de un fluoruro de calcio plano-convexo o lentes antireflejos ZnSe recubierto (infrarrojos productos ópticos, Farmingdale, NY) en su no-grabado final. Protegidos espejos de oro (PF10-03-M01, Thorlabs, Newton, Nueva Jersey) se utilizan para dirigir el rayo láser infrarrojo medio en el anillo de enfoque. </li></ol> 2. Electrospray configuración <ol><li> El sistema de electrospray se pueden construir con los siguientes componentes: la unión de metal con puntera perfluoroelastómero conductores, accesorios, manga tubos, agujas puerto (por ejemplo, U-435, M215, M-331Nx, F-242x, 9013, respectivamente IDEX Salud y Ciencias, Oak Harbor, WA), así como tubos de sílice fundido (por ejemplo, CT360-100-50-5, Nuevo Objetivo Inc., Woburn, MA). El emisor se recomienda es de acero inoxidable y tiene una punta cónica con un identificador de 50 micras (por ejemplo, MT320-50-5-5, Nuevo Objetivo Inc., Woburn, MA). </li><li> Prepare solución al 50% de metanol con 0,1% de ácido acético (v / v) para el electrospray. La solución es bombeada a un ritmo de 200 a 300 nl / min por una bomba de jeringa (Harvard Apparatus, Holliston, MA), utilizando, por ejemplo, un. Jeringa 500 l (81 222, la empresa Hamilton, Reno, NV) </li><li> Aplicar alto voltaje entre 2.800 y 3.000 V para la unión de metales por una fuente de alimentación regulada (PS350, Stanford Research Systems Inc, Sunnyvale, CA) para generar un electrospray estable. Asegúrese de que todas las conexiones eléctricas estén seguras y protegidas con el blindaje a tierra. El contacto directo con alto voltaje descubierto puede provocar una descarga eléctrica. </li></ol> 3. De manipulación de muestras <ol><li> Obtener el tejido vivo o una muestra de células de una fuente apropiada y mantenerlos lo más recomendable antes del análisis. El Allium cepa bombillas para esta demostración fueron comprados en una tienda local. Cortar una bombilla longitudinalmente por un bisturí quirúrgico. Un segmento de unos pocos centímetros de la escala se seccionó en una tira. Una monocapa intacta del tejido epidérmico interior se despega. </li><li> La superficie húmeda de la epidermis se usa para montar el tejido sobre un portaobjetos de microscopio de vidrio limpiado previamente. Sostenga el deslizador por el titular de la placa de propósito general (FP01, Thorlabs Inc., Newton, Nueva Jersey) montado en una etapa de traducción de tres ejes para el posicionamiento. Adquisición de los espectros de masas inmediatamente después del montaje de la muestra para evitar la degradación de la célula. </li></ol> 4. Sistema de visualización <ol><li> Dos microscopios de larga distancia se utilizan para seleccionar las células para su análisis y para mantener la distancia entre la punta de la fibra afilada y la superficie celular. Éste está montado en ángulo de 15 ° y 30 ° medidos desde la superficie. Este sistema consiste en aldistancia ong microscopio de vídeo (7x zoom óptico de precisión, Edmund Optics, Barrington, NJ), con una infinidad de 5x lente con corrección objetiva (Apo M Plan de 5x, Mitutoyo Co., Kanagawa, Japón) equipado con una cámara CCD (Marlin F131, Allied Vision Technologies , Stadtroda, Alemania) para el seguimiento y captura de imágenes. </li><li> El microscopio segundo video está montado en un ángulo recto con la superficie de la muestra para visualizar las células de la parte superior. Este sistema proporciona una retroalimentación visual para alinear el extremo de la fibra sobre la celda seleccionada para la ablación y el análisis. Se trata de un zoom óptico 7x precisión (Edmund Optics, Barrington, NJ), equipado con una infinidad de 10x corregido largo lente objetivo de trabajo a distancia (M Plan de 10x Apo, Mitutoyo Co., Kanagawa, Japón) y una cámara CCD. </li></ol> 5. Adquisición de espectros de masas <ol><li> Encienda el láser, electrospray y el espectrómetro de masas, por ejemplo, un sistema de cuadrupolo-tiempo de vuelo (Q-TOF Premier, Waters, Milford, MA). Optimizar la geometría de la fuente de iones LAESI ajustando la posición del punto de ablación y el emisor electrospray respecto a la otra y el orificio del espectrómetro de masas para lograr la intensidad máxima de iones. </li><li> Seleccione la celda de interés para el análisis utilizando el sistema de visualización de primera vista. Después de la selección, mover el escenario de la muestra para que la celda seleccionada directamente por debajo de la punta afilada de la fibra óptica. Con el microscopio vista lateral reajustar la punta-a-distancia de la superficie celular de aproximadamente 20 micras. </li><li> Fuego pulsos de láser en la celda seleccionada a través de la punta de la fibra grabado. Este resultado en la perforación de la pared celular y la expulsión del citoplasma en forma de partículas. Después de postionization por el electrospray, los iones producidos son recogidos por el espectrómetro de masas y los espectros de masas se registran. </li><li> Después de la adquisición de datos, puesto que el láser, electrospray y el espectrómetro de masas en modo de espera o apagarlos. Mantenga la ablación A. cepa muestra de tejido para su observación al microscopio óptico opcional. </li></ol> 6. Resultados representante Éxito de la ablación de una sola célula en los resultados de la explosión de la pared celular y la colección de un espectro de masas LAESI. Un experimento sin éxito por lo general da lugar a ninguna de ablación y / o no hay un espectro de masas. Para fines de demostración, se presentan los resultados del análisis LAESI-MS de una sola célula epidérmica incrustado de una A. cepa del bulbo. La figura 2a muestra una imagen representativa de microscopía óptica tras la toma de muestras de una célula. La pared celular es perforada y el alcance de la perforación está limitada por las fronteras con las células vecinas. Las células adyacentes parecen estar intactas. La Figura 2b muestra un espectro representativo LAESI producidos en masa de la célula. Una gran variedad de iones se detectan a partir de la A. cepa de células y en base a sus masas precisas, patrones de isótopos de distribución, y, en algunos casos, los espectros de masas en tándem, que son asignados provisionalmente a metabolitos endógenos. Los iones detectados a partir de una sola célula fueron similares a los observados en el análisis del tejido mismo convencionales LAESI-MS de varias celdas. 13 <img src="/files/ftp_upload/2144/2144fig1.jpg" alt="Figura 1" /> Figura 1. Esquemática de análisis de células individuales por LAESI-MS. Un pulso de láser infrarrojo medio se une en una fibra óptica y entregado a la muestra colocada en el portaobjetos de vidrio. La afilada punta de la fibra se concentra la luz en la celda de destino y la energía depositada estalla la célula. La pluma de ablación producido (puntos rojos) se combina con una pluma de electrospray (puntos negro) que las gotas se unen (puntos verdes) que resulta en la producción de iones de la muestra específica. Estos iones son analizadas y detectadas por el espectrómetro de masas (MS). Dos microscopios de vídeo larga distancia, un microscopio y el microscopio 2, se utilizan para controlar la distancia entre la punta de la fibra y la superficie de la célula y para seleccionar una celda para el análisis, respectivamente. <img src="/files/ftp_upload/2144/2144fig2a.jpg" alt="Figura 2a" /> Figura 2a. Ablación marca en una sola célula epidérmica de una A. cepa del bulbo. Las células adyacentes no presentan daños visibles y, posteriormente, se pueden analizar por separado. <img src="/files/ftp_upload/2144/2144fig2b.jpg" alt="Figura 2b" /> Figura 2b. Masa correspondiente a la ablación de un solo espectro A. cepa de células epidérmicas muestra la presencia de metabolitos primarios y secundarios.

<ol>
	<li>Cooks, R. G., Ouyang, Z., Takats, Z., Wiseman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Mass+Spectrometry.">Ambient Mass Spectrometry.</a> Science. 311, 1566-1570 (2006).</li><li>Chen, Z. Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+plume+expansion+in+atmospheric+pressure+midinfrared+laser+ablation+of+water-rich+targets.">Early plume expansion in atmospheric pressure midinfrared laser ablation of water-rich targets.</a> Physical Review E. E77, 036316-036316 (2008).</li><li>Li, Y., Shrestha, B., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Molecular+Imaging+by+Infrared+MALDI.">Atmospheric Pressure Molecular Imaging by Infrared MALDI.</a> Mass Spectrometry. Analytical Chemistry. 79, 523-532 (2006).</li><li>Li, Y., Shrestha, B., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Infrared+MALDI+Imaging+Mass+Spectrometry+for+Plant+Metabolomics.">Atmospheric Pressure Infrared MALDI Imaging Mass Spectrometry for Plant Metabolomics.</a> Analytical Chemistry. 80, 407-420 (2007).</li><li>Nemes, P., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Ablation+Electrospray+Ionization+for+Atmospheric+Pressure+in+Vivo,+and+Imaging+Mass+Spectrometry.">Laser Ablation Electrospray Ionization for Atmospheric Pressure in Vivo, and Imaging Mass Spectrometry.</a> Analytical Chemistry. 79, 8098-8106 (2007).</li><li>Shrestha, B., Li, Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+pharmaceuticals+and+excreted+xenobiotic+and+endogenous+metabolites+with+atmospheric+pressure+infrared+MALDI+mass+spectrometry.">Rapid analysis of pharmaceuticals and excreted xenobiotic and endogenous metabolites with atmospheric pressure infrared MALDI mass spectrometry.</a> Metabolomics. 4, 297-311 (2008).</li><li>Nemes, P., Barton, A. A., Li, Y., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+Molecular+Imaging+and+Depth+Profiling+of+Live+Tissue+by+Infrared+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Ambient Molecular Imaging and Depth Profiling of Live Tissue by Infrared Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 80, 4575-4582 (2008).</li><li>Nemes, P., Barton, A. A., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+of+Metabolites+in+Tissues+under+Ambient+Conditions+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Three-Dimensional Imaging of Metabolites in Tissues under Ambient Conditions by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 81, 6668-6675 (2009).</li><li>Nemes, P., Woods, A. S., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Imaging+of+Small+Metabolites+and+Lipids+in+Rat+Brain+Tissues+at+Atmospheric+Pressure+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">Simultaneous Imaging of Small Metabolites and Lipids in Rat Brain Tissues at Atmospheric Pressure by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 82, 982-988 (2010).</li><li>Shrestha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+lipids+and+small+metabolites+in+mouse+brain+tissue+by+AP+IR-MALDI+and+reactive+LAESI+mass+spectrometry.">Direct analysis of lipids and small metabolites in mouse brain tissue by AP IR-MALDI and reactive LAESI mass spectrometry.</a> Analyst. , (2010).</li><li>Sripadi, P., Nazarian, J., Hathout, Y., Hoffman, E., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+analysis+of+metabolites+from+the+untreated+tissue+of+Torpedo+californica+electric+organ+by+mid-infrared+laser+ablation+electrospray+ionization+mass+spectrometry.">In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry.</a> Metabolomics. 5, 263-276 (2009).</li><li>Shrestha, B., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Metabolic+Profiling+of+Single+Cells+by+Laser+Ablation+Electrospray+Ionization+Mass+Spectrometry.">In Situ Metabolic Profiling of Single Cells by Laser Ablation Electrospray Ionization Mass Spectrometry.</a> Analytical Chemistry. 81, 8265-8271 (2009).</li><li>Shrestha, B., Nemes, P., Vertes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+and+Analysis+of+a+Single+Cell+by+Consecutive+Laser+Pulses.">Ablation and Analysis of a Single Cell by Consecutive Laser Pulses.</a> Applied Physics A. , (2010).</li></ol>

El contenido de agua del citoplasma celular y la resistencia a la tracción de la pared o membrana celular son factores fundamentales que rigen la ablación de las células individuales en LAESI-MS análisis. La fluencia del láser tiene que ser ajustada para alcanzar los umbrales de la ablación de los diferentes tipos de células. La fluencia del láser entrega depende de la energía del pulso láser y de la distancia entre la punta de la fibra y la superficie celular. Minimizar la perturbación de las células vecinas durante el análisis de células individuales es crítico. Mantener la energía del láser en el umbral de ablación limita el efecto del análisis de las células adyacentes. Una de las posibles aplicaciones es usar las células individuales, como los píxeles naturales volumétrico (voxels) para la industria química de imágenes por la EM. En comparación con la recopilación de datos en una cuadrícula rectangular artificial a menudo, de imagen química de un tejido de una célula a la vez es más probable que mantener la organización espacial de las interacciones bioquímicas entre las células.

<!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>Acetic acid</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>45727</td>
<td>&nbsp;</td>
<tr>
<td>Methanol</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>65548</td>
<td>&nbsp;</td>
<tr>
<td>1-methyl-2-pyrrolidinon</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>M79603</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>39259</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

direct analysis of single cells by mass spectrometry at atmospheric pressure

Análisis de productos bioquímicos en las células individuales es importante para entender el metabolismo celular, ciclo celular, la adaptación, los estados de enfermedad, etc Incluso los mismos tipos de células presentan distribución heterogénea bioquímicos en función de sus condiciones fisiológicas y la interacción con el medio ambiente. Los métodos convencionales de espectrometría de masas (MS) que se utiliza para el análisis de biomoléculas en células individuales se basan en la preparación de muestras amplias. La eliminación de las células de su entorno natural y de procesamiento de muestras extensas podría conducir a cambios en la composición celular. Métodos de ionización ambiental permitirá el análisis de muestras en su medio ambiente y sin preparación de la muestra amplia. 1 Las técnicas basadas en el infrarrojo medio (infrarrojo medio), la ablación por láser de materiales biológicos en longitud de onda de 2,94 micras utilizan la excitación repentina de agua que los resultados en la fase explosión. 2 técnicas de ionización ambiental sobre la base de la radiación láser infrarrojo medio, como la ionización electrospray ablación con láser (LAESI) y la presión atmosférica de infrarrojos láser asistida por matriz de ionización de desorción (AP-MALDI IR), han demostrado con éxito la capacidad de analizar directamente el agua tejidos ricos y biofluidos a presión atmosférica. 3-11 En LAESI la pluma láser infrarrojo medio de ablación que se compone principalmente de partículas neutrales de la muestra se funde con las gotas de electrospray muy cargado para producir iones. Recientemente, a mediados de IR ablación de las células individuales se realizó mediante la entrega de la radiación de infrarrojo medio a través de una fibra de grabado. La columna de humo generada a partir de esta ablación fue postionized por un electrospray permitiendo el análisis de metabolitos diferentes en las células individuales por LAESI MS-12. En este artículo se describe el protocolo detallado para el análisis de células individuales con LAESI-MS. El video presentado demuestra el análisis de una única célula epidérmica de la piel de un bulbo de Allium cepa. El esquema del sistema se muestra en la Figura 1. Un ejemplo representativo de la ablación de células individuales y un espectro de masas LAESI de la célula se proporcionan en la Figura 2.

Watch this Scientific Journal Video about Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure at JoVE.com

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Análisis de las células individuales se realiza por espectrometría de masas en las células animales y vegetales a la presión atmosférica mediante el uso de una fibra óptica para afilado de la muestra de las células de ionización electrospray ablación con láser (LAESI) espectrometría de masas.

El análisis directo de las células individuales mediante espectrometría de masas a presión atmosférica

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

direct-analysis-single-cells-mass-spectrometry-at-atmospheric

Research

JoVE Journal

Biology

15.9K vistas.  George Washington University. Análisis de las células individuales se realiza por espectrometría de masas en las células animales y vegetales a la presión atmosférica mediante el uso de una fibra óptica para afilado de la muestra de las células de ionización electrospray ablación con láser (LAESI) espectrometría de masas.

Video: Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI preparación de la muestra: el método de ultra capa fina

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Investigación

Biología

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

El análisis de grandes complejos de proteínas por espectrometría de masas estructurales

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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. ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Presión atmosférica de Imagen Molecular de los tejidos biológicos y biopelículas por LAESI Espectrometría de Masas

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amida de hidrógeno / deuterio intercambio y MALDI-TOF análisis de espectrometría de masas de Pak2 activación

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análisis de RNA Global Síntesis en el nivel de células individuales siguiente Hipoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Análisis de retrotransposition LINE-1 en el Núcleo Un piso

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Aplicaciones de la sonda: Single-Imaging espectrometría de masas y análisis de celda única bajo condiciones ambientales

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Preparación de muestras para análisis de proteómica basado en la espectrometría de masa de microvasos Ocular

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...