Este trabajo es apoyado en parte por los Institutos Nacionales de Salud Grant GM-26738 (a JAT).

1. Pak2 activación <ol><li> Añada la cantidad de pre-prueba de la caspasa 3 a 20 l de 12 mg / ml Pak2 en un buffer y (50 mM de TDT mM Tris-HCl, 150 mM NaCl, y 2 a pH 7,5) se incuba a 34 ° C durante 30 minutos totalmente a romper, y Pak2 activar. </li><li> Analizar los productos de degradación por SDS-PAGE (10%) y la tinción Coommassie azul. </li><li> Agregue el Pak2 se pegaba a la activación de amortiguación B (50 mM Tris-HCl, 150 mM NaCl, 15 mM MgCl 2, 10 mM ATP) a un pH de 7,5, en 20 l para una concentración final de 10 mg / ml Pak2, e incubar a 34 ° C durante 30 minutos para activar plenamente Pak2 por autofosforilación en 8 sitios. </li><li> Confirmar la ruptura completa de Pak2 por la migración de la p34 y p27 fragmentos según lo determinado por SDS-PAGE 10. Un adecuado control de las condiciones de la autofosforilación se hace a través de autorradiografía en un experimento separado con (32P) ATP o la espectrometría de masas para detectar fosfopéptidos. </li></ol> 2. Identificaciónde la pepsina-digeridos Pak2 Fragmentos <ol><li> Añadir 1 ml de 0,1% de ácido Trifluroacetic (TFA) a un pH de 2,5 a lavar las cuentas de agarosa dos veces antes de su uso. A continuación, centrifugar las muestras de 15 segundos a 2.000 xg para extraer las bolas. </li><li> Añadir Pak2 (20 mg en 2 l de 150 mM NaCl, 50 mM de TDT mM Tris-HCl y 2 a pH 7,5) a 100 l de TFA al 0,1%, e incubar la muestra durante 5 minutos con 30 l de pepsina conjugado bolas de agarosa . </li><li> Equilibrar una columna de HPLC en fase reversa C18 con un sistema solvente principal de 0,1% TFA a pH 2,5. El resumen de la pepsina (50 l) se eluye con un lineal de 100 min gradiente de 0 a 80% de acetonitrilo que contiene 0,1% de TFA en un caudal de 0,2 ml / min. Fracciones se recogen cada 2 minutos. </li><li> Seco de las fracciones HPLC con un Speed-Vac (3 h) y volver a suspender las muestras en una solución de acetonitrilo (5 l) y 0,1% de TFA (5 l) a pH 2,5. </li><li> Inicialmente, cada fracción se analiza para detectar péptidos utilizando el MALDI-TOF PerSeptive Biosystems Voyager STR DE (PE Biosystems, Foster City, CA, EE.UU.). </li><li> El 16 fracciones que contiene péptidos son sometidos a espectrometría de masas en tándem con un espectrómetro de masas Q-TOF y QSTAR XL oMALDI MS / MS para identificar los fragmentos. </li><li> La secuencia de cada péptido se verifica por los iones de producto generado por la espectrometría de masas en tándem con el teórico m / z de coeficientes basados ​​en la secuencia primaria de Pak2 utilizar el sitio de proteínas Prospector ( <a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ) 11. </li></ol> 3. Medición de la Amida H / D intercambio <ol><li> Mantenga todas las soluciones y reactivos necesarios para H / D intercambio a 25 ° C en un baño de agua. ATP se disuelve en agua y el pH se ajustó a 7,0 antes de su uso. Los otros 4 mM de ATP en el buffer D 2 O es evitar ATP disociada de Pak2 después de la dilución. </li><li> Iniciar H / D intercambio mediante la adición de 18 l de tampón D 2 O (50 mM MOPS pH 6.98, 125 mM NaCl) a 2 l (20 mg) De Pak2 en un tampón que contiene 150 mM NaCl, 50 mM de TDT mM Tris-HCl (pH 7.5) y 2, resultando en un nivel Pak2 de 10 mg / ml y un pH de 7,0 a 0 ° C. </li><li> Incubar las muestras por triplicado en el intercambio H / D de 0, 0,5, 1, 3, 5 y 10 minutos, o 24 h. </li><li> Apagar el proceso de H / D intercambio mediante la adición de 180 l de helado de TFA 0,1% a un pH de 2,2, lo que eleva el pH a 2,5 en un volumen final de 200 microlitros. Para las muestras que contienen ATP, 180 l de TFA helada 0,11% a un pH de 2,2 se utiliza para mantener el pH a 2,5 en la condición de temple. </li><li> Añadir un volumen (100 l) de cada muestra apagado a 30 l de la pepsina activa conjugado bolas de agarosa. La digestión de la pepsina se le permite continuar en el hielo durante cinco minutos y las muestras se agitaron cada 30 segundos. </li><li> Terminar la digestión por centrifugación de la muestra durante 15 segundos a 5000 xg a 4 º C para eliminar la pepsina. </li><li> Rápidamente congelar las muestras en N2 líquido y se almacena a -80 ° C durante no más de 2días antes de MALDI-TOF análisis. </li><li> La matriz de MALDI es de 5 mg / ml de α-ciano-4-hidroxicinámico (CHCA) disuelto en una solución (1:1:1) de acetonitrilo, etanol y 0,1% de TFA a pH 2,5 y se mantiene a 0 º C. </li><li> Parcialmente descongelar cada muestra de forma rápida y mezclar 1 l de cada muestra con una l de la matriz y el lugar en un 4 º C MALDI placa objetivo. </li><li> Aplicar un vacío moderado a secar la placa en los puntos 30 segundos antes de MALDI-TOF análisis. El tiempo entre la descongelación de la muestra y la recuperación de espectro es de unos 3 min. </li><li> Adquirir MALDI-TOF espectros de masas con un PerSeptive STR Biosystems Voyager DE. La adquisición de datos a una velocidad de muestreo de 2 GHz a 100.000 canales de datos, con una tensión de 20.000 V la aceleración, y una tensión de red 65% mediante la extracción de retraso con un retraso de pulso de 180 ns. Un centenar de las exploraciones se hacen un promedio de 2 min. El instrumento MALDI-TOF no se limita a cualquier marca o cualquier otro software en particular. </li><li> Use el Explorador de datos del programa informático de 4,0 a calculse comió la deuteración de cada fragmento. </li><li> Calcule el cambio de nuevo sobre la base de la relación entre el número real de deuterón incorporado a las 24 horas de H / D intercambio dividido por todos los sitios posibles canjeables, que son las amidas de columna vertebral, excepto la amida N-terminal (Figura 1). </li></ol> 4. Los resultados representativos: La escisión de la caspasa y la autofosforilación son verificados por SDS-PAGE tinción Commassie. Pak2 inactiva es una sola banda de 58 kDa. División de la caspasa Pak2 es completa, y produce dos fragmentos, p27 y p34 que migran por separado. La migración de p27 y p34 autophosphorylated muestra una superposición en el gel debido a la migración retardada de la completamente fosforilados p27 fragmento (Figura 2). H / D intercambio experimentos se llevan a cabo varias veces entre 0 y 10 min (Figura 3). A modo de ejemplo, el curso temporal de la incorporación de deuterio en el m / zpEAK 1697.85 de Pak2 inactiva está compuesta de múltiples picos isotópicos como lo muestra el desplazamiento de la masa sobre el paso del tiempo. <img alt="Figura 1" src="/files/ftp_upload/3602/3602fig1.jpg" /> Figura 1. La ecuación para el cálculo de la deuteración. <img alt="Figura 2" src="/files/ftp_upload/3602/3602fig2.jpg" /> Figura 2. Autofosforilación y la escisión de la caspasa Pak2. Caspasa-escindida Pak2 (carril izquierdo), Pak2 intacta inactivo (carril central) y la caspasa-escindida autophosphorylated Pak2 (carril derecho) fueron analizados por SDS-PAGE y tinción Coommassie azul. Adaptado de Hsu, YH et al 2. <img alt="Figura 3" src="/files/ftp_upload/3602/3602fig3.jpg" /> Figura 3. Espectro de masas de un curso de tiempo de la incorporación de deuterio, para Pak2 inactivo. Pak2 inactiva fue sometido a H / D exchaESN de 00 a 10 min y se analizaron por MALDI-TOF. Un ejemplo de la amplia distribución isotópica de MALDI-TOF de pico m / z 1697.85 durante el curso temporal de H / D intercambio se muestra. La línea roja punteada indica el promedio de la envolvente de la masa y el desplazamiento de la masa sobre la muestra deuteración de un péptido.

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 283, 9820-9827 (2008).</li><li>Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hPAK65+by+caspase+cleavage+induces+some+of+the+morphological+and+biochemical+changes+of+apoptosis.">Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13642-13647 (1997).</li><li>Rudel, T., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+morphological+changes+in+apoptotic+cells+regulated+by+caspase-mediated+activation+of+PAK2.">Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.</a> Science. 276, 1571-1574 (1997).</li><li>Roig, J., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytostatic+p21+G+protein-activated+protein+kinase+gamma-PAK.">Cytostatic p21 G protein-activated protein kinase gamma-PAK.</a> Vitam. Horm. 62, 167-198 (2001).</li><li>Walter, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+and+activation+of+p21-activated+protein+kinase+gamma-PAK+by+CPP32+(caspase+3).+Effects+of+autophosphorylation+on+activity.">Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.</a> J. Biol. Chem. 273, 28733-28739 (1998).</li><li>Clauser, K. R., Baker, P., Burlingame, A. 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No hay conflictos de interés declarado.

Identificación de los fragmentos digeridos con pepsina es un paso crítico de la experiencia de H / D intercambio. Una incorrecta identificación de los péptidos puede llevar a una conclusión equivocada. Pak2 pepsina digerida se eluye a través de una columna HPLC en fase reversa C18 para obtener un fondo limpio. En nuestros experimentos, un total de 40 fracciones fueron recogidos y sometidos a MALDI-TOF. Múltiples péptidos podrían ser identificados en cada una de las fracciones. Los péptidos fueron sometidos a espectrometría de masas tándem (Q-TOF MS / MS y oMALDI MS / MS) para identificar sus secuencias. Los espectros MS / MS de un péptido debe tener al menos tres iones producto para confirmar la identificación de los péptidos. El Explorador de Datos programa de ordenador 4.0 (Applied Biosystems) realiza la corrección de línea de base inicial y filtración de ruido. Cada espectro deberá ser calibrado a partir de dos picos en secuencia. Calibramos nuestro espectro de la masa teórica de la undeuterated m / z que son 923,45 y 1.697,84. Tél precisión la masa después de la calibración puede llegar a 10 ppm. Al deuteración, el mono-isótopo puede cambiar a una masa mayor. Aumenta unitaria paso a estas relaciones m / z dado a las masas asociadas a una mayor deuteración. El pico de intensidad de la envoltura de masa no afectará el nivel de deuteración. Sin embargo, las intensidades de los picos isotópicos en un sobre de masas en particular son esenciales para el cálculo de la masa media. El primer paso para el cálculo del deuterón incorporado es restar la masa de péptidos de la muestra no deuterado de la muestra deuterado. Hay otros tres factores, el D 2 O de dilución, los deuterones residual en las cadenas laterales y el intercambio de nuevo, tendrá que ser consideradas en el cálculo (Figura 1). La dilución D 2 O es el factor de dilución de D 2 O después de mezclar la muestra y D 2 O de amortiguación para iniciar el intercambio H / D. El deuterón residual (4,5%) en las cadenas laterales de la pepsina de digerirpéptidos ed tiene que restarse. La parte posterior de intercambio es la inevitable pérdida de deuteración en todos los péptidos deuterado y la pepsina digerida en el proceso de medición de la masa. El péptido más accesible disolvente (m / z = 1105,60) después de 24 horas de H / D intercambio representa una región deuteración completo. El número deuterón incorporados para cada uno de los péptidos es la diferencia entre el centro de gravedad de los péptidos péptica deuterados y no deuterados-Pak2. Los más deuterado péptido m / z 1105 fue seleccionado para representar deuteración completa cuando Pak2 fue a las 24 horas de H / D intercambio. La Ecuación de Canje Volver fue calculado por el número real de deuterio incorporado a las 24 horas de H / D intercambio dividido por todos los sitios posibles canjeables en péptido m / z 1105.

<table border="1"></table><table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> D 2 O </td><td> Aldrich </td><td> 7789-20-0 </td><td></td></tr><tr><td> α-ciano-4-hidroxicinámico </td><td> Sigma </td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI-TOF </td><td> PE Biosystems </td><td> Voyager DE STR </td><td></td></tr><tr><td> Q-TOF espectrómetro de masas </td><td> Q-TOF Ultima-Global </td><td></td><td></td></tr><tr><td> QSTAR XL oMALDI MS / MS </td><td> Applied Biosystems </td><td></td><td></td></tr><tr><td> Data Explorer 4.0 programa de ordenador </td><td> Applied Biosystems </td><td></td><td></td></tr&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

Amida de hidrógeno / deuterio intercambio (H / D intercambio) acoplada a espectrometría de masas ha sido ampliamente utilizado para analizar la interfaz de las interacciones proteína-proteína, los cambios conformacionales de proteínas, dinámica de las proteínas y las interacciones ligando-proteína. H / D intercambio de las posiciones de columna vertebral amida se ha utilizado para medir las tasas de deuteración de las micro-regiones de una proteína por espectrometría de masas 1,2,3. La resolución de este método depende de la digestión con pepsina de la proteína de interés deuterado en péptidos que normalmente rango 3-20 residuos. Aunque la resolución de H / D intercambio medido por espectrometría de masa es menor que la resolución solo residuo medido por el heteronuclear único coherencia cuántica (HSQC) el método de RMN, espectrometría de masas de la medición de H / D intercambio no estará limitado por el tamaño de la proteínas 4. H / D intercambio se lleva a cabo en una solución acuosa que mantiene la conformación de proteínas. Ofrecemos un método que utilriza el MALDI-TOF para la detección de dos, en lugar de un sistema HPLC / ESI (ionización electrospray)-MS 5,6. El MALDI-TOF ofrece datos precisos sobre la intensidad de la masa de los péptidos de la proteína digerida, en este caso quinasa Pak2 proteína (también llamada γ-Pak). La proteólisis de Pak 2 se lleva a cabo en una digestión de la pepsina en línea. Este método alternativo, cuando el usuario no tiene acceso a una columna de HPLC y la pepsina conectado a la espectrometría de masas, o cuando la columna de la pepsina en HPLC no se traduce en un mapa de una digestión óptima, por ejemplo, la muy unidas por puentes disulfuro secreta fosfolipasa A 2 (sPLA2). Utilizando este método, hemos logrado controlar los cambios en el nivel deuteración durante la activación de la caspasa 3 Pak2 por división y la autofosforilación 7,8,9.

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

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

MALDI-TOF espectrometría de masas fue utilizada con éxito para controlar la amida hidrógeno / deuterio cambio en la activación de proteína quinasa Pak2.

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 ...

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17.7K vistas.  Tunghai University. MALDI-TOF espectrometría de masas fue utilizada con éxito para controlar la amida hidrógeno / deuterio cambio en la activación de proteína quinasa Pak2.

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

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. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

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 ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Creación de perfiles proteoma redox tiol Uso del etiquetado de espectrometría de masas

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

El uso de MALDI-TOF espectrometría de masas y una base de datos personalizada para caracterizar bacterias indígenas a una cueva entorno único (Kartchner Cavernas, AZ, EE.UU.)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Reacción seleccionada Monitoreo espectrometría de masas para Absolute Proteína cuantificación

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Enriquecimiento de la Matriz Extracelular Las proteínas de los tejidos y la digestión en péptidos para el análisis de espectrometría de masas

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

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 ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flujo de trabajo completo para el análisis de las histonas modificaciones post-traduccionales El uso de espectrometría de masas de abajo hacia arriba: A partir de la histona Extracción de Análisis de Datos

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...