Este trabajo fue financiado por el Instituto Nacional del Cáncer Clínica Proteómica del Centro de Tecnología de Evaluación (CPTAC) beca (# U24 CA126476), así como una beca de la Industria del Entretenimiento Foundation (EIF) y el Fondo de las Mujeres del FEI de Investigación del Cáncer en el Consorcio el cáncer de mama el descubrimiento de biomarcadores y las generosas donaciones de la Fundación Keck, de la Fundación Canarias, y la Paul G. Allen Family Foundation.

Procedimiento experimental: El ensayo requiere péptidos sintéticos y anticuerpos anti-péptido. Péptidos seleccionados deben ser exclusivos de la proteína de interés, contienen entre 8 y 22 aminoácidos, y no se conocen modificaciones post-traduccionales. Los residuos de metionina generalmente se evitan y los péptidos que contienen aminoácidos dibásicos (por ejemplo, KK, KR, RR) no son deseables. Para esta técnica, es común el uso de péptidos de isótopos estables etiquetados como estándares internos, la incorporación de pesados ​​(13 C y N 15) aminoácidos marcados en el C-terminal del péptido (es decir, K o R etiqueta). El siguiente protocolo describe un ensayo para medir el GDSLAYGLR péptido de la osteopontina proteína de ratón, utilizando anticuerpos anti-péptido obtenido de Epitomics Inc. (Burlingame, CA) y péptidos sintéticos de Nueva Inglaterra Péptidos (Gardner, MA). El protocolo consta de tres pasos principales (Figura 1): 1) la digestión de tripsina de la mezcla compleja de proteínas, 2) Enriquecimiento de péptidos 3) El análisis por espectrometría de masas. Se demostrará en una muestra de plasma humano enriquece con la proteína osteopontina ratón. 1. Tripsina digestión enzimática y la limpieza <ol><li> Descongele 10 l alícuota de plasma limpia en hielo. </li><li> Determinar la concentración de proteínas totales mediante el ensayo de BCA y centrifugar la muestra para eliminar los sólidos en suspensión. </li><li> Pipetear 10 l alícuota de su tubo de almacenamiento a una placa de 1000 l pozos profundos y cubrir con Pierce-capaz de película. </li><li> Añadir 20 l de 9 millones de nuevos urea / 30 mM ditiotreitol (DTT) (6M final de la concentración de urea / 20 mM DTT) a cada muestra. </li><li> Incubar durante 30 minutos a 37 ° C. </li><li> Añadir 3 l de 500 mM yodoacetamida fresca (final IAM 50 mm) a cada muestra. </li><li> Incubar durante 30 minutos en la oscuridad a temperatura ambiente. </li><li> Añadir 257 l de 100 mM Tris (pH 8) (urea diluye a ~ 0,6 M). </li><li> Añadir 10 l de solución madre de tripsina (1 mg / l; de 1:50 enzima: sustrato de relación). </li><li> Incubar 37 ° C durante la noche (12-16 horas). </li><li> Añadir 3 l de ácido fórmico puro (concentración final de 1%). </li><li> Añadir estándar de isótopos estables (normas se agregan varias si se realiza un ensayo multiplex, por lo general se trata de 10 l que contiene 50-100 fmol de péptido estándar marcados con isótopos). </li><li> Lavar la placa cartucho de Oasis y con 500 l de 0,1% de ácido fórmico en acetonitrilo 80%, descartando el flujo continuo. Repita esto 3 veces. </li><li> Equilibre la placa del cartucho mediante la adición de 500 l de 0,1% de ácido fórmico en agua, y descartar el filtrado. Repita este 4 veces. </li><li> Cargar las muestras de digerir a la placa del cartucho y ajustar el vacío para que el flujo es muy lento. </li><li> Lavar con 500 l de 0,1% de ácido fórmico en agua, y descartar el filtrado. Repita esto 3 veces. </li><li> Péptidos eluir mediante la adición de 2 x 500 l de 0,1% de ácido fórmico en acetonitrilo al 80% en 1000 l profunda y placa (no se descarta el flujo a través). </li><li> Liofilizar (o speedvac) el eluyente hasta desecación. (La liofilización es el método preferido) </li><li> Reconstituir seca péptidos mediante la adición de 50 l de PBS + 0,03% CHAPS. </li></ol> 2. Péptido de enriquecimiento de inmunoafinidad <ol><li> Transferir la muestra a nivel Kingfisher placas de 96 pocillos. </li><li> Añadir un anticuerpo mg y 1,5 l proteína G-magnéticas recubiertas por objetivo (que es opcional para entrecruzar los anticuerpos a las bolas antes de la adición). Asegúrese de cuentas son también suspendido por temblor o agitación. </li><li> Cubrir la placa con papel de aluminio. </li><li> Incubar toda la noche (16.12 h) con suave volteretas para asegurarse de cuentas son suspendidas. </li><li> Centrifugar la placa a 32 xg durante 5 segundos para eliminar el líquido de la superficie de la lámina. </li><li> Quite la cubierta de aluminio. </li><li> Lavado y la elución de las cuentas se puede realizar manualmente o de forma automatizada. Este procedimiento describe los pasos automatizados en una plataforma de Kingfisher. </li><li> Lavar las perlas de dos veces con 200 l de PBS + 0,03% CHAPS (1 minuto por lavado). </li><li> Lavar las perlas de una vez con 200 l de dilución de 1:10 de PBS + 0,03% CHAPS (1 minuto). </li><li> Los péptidos se eluyeron en 25 l de ácido acético al 5% + 0,03% CHAPS. </li><li> Coloque la placa de elución de un imán y la transferencia que el efluente de una placa y 96, teniendo cuidado de no transferencia de las cuentas de sobra. </li><li> Cubrir la placa con una estera de sellado. </li><li> La placa que contiene el efluente se entrega el espectrómetro de masas de triple cuadrupolo para el análisis. </li></ol> 3. Análisis mediante el control de reacción múltiple - espectrometría de masas <ol><li> Transiciones de SRM / MRM análisis pueden ser seleccionados y optimizados mediante la infusión de una solución picomole 1 por microlitro de péptido estándar en acetonitrile/0.1% al 30% de ácido fórmico en el espectrómetro de masas de 0,5 l / min. Una vez rociado estable se obtiene, recoge un espectro MS / MS. </li><li> Las transiciones son seleccionados de todo el espectro MS / MS mediante la identificación de three iones abundantes fragmentos en una región del espectro, con poco ruido. Para multiplicar cargado de iones de péptidos, los fragmentos y de iones detectados en m / z&gt; precursor m / z son generalmente las mejores para el desarrollo de ensayos de SRM / MRM. La figura 2 muestra el espectro MS / MS y otros iones de transición 476,3&gt; 508,3, 579,3 , 692.4 (transiciones para el pesado de isótopos estables estándar 481.3 no&gt; 518,3, 589,3, 702,4 se muestra) para la GDSLAYGLR péptido. </li><li> Dependiendo de la marca y modelo del espectrómetro de masa utilizado, hay varios parámetros que pueden ser optimizados para cada transición. Aquí, hemos optimizado la energía de colisión de cada transición seleccionada por la rampa de la energía de la colisión y el control del nivel de señal. Una energía de colisión de 25 se utilizó para cada transición. </li><li> En pocas palabras, una configuración típica de SRM / MRM análisis es el siguiente: Fase móvil (A) de ácido fórmico al 0,1%, (B) acetonitrilo 90% / 0,1% de ácido fórmico, 0,3 x 5 mm de columna trampa de C18, 75 ID m x 15 cm C18 analítica (Reprosil-Pur C18 AQ, 120 A poro °). El volumen de inyección es de 10 l, y la muestra se carga durante 5 minutos a 3% B con un caudal total de 3 l / min y se eluyó con un gradiente lineal de 3-45% B en 300 a 400 nL / min en 10 min. Condiciones en un QTRAP 4000 (ABSCIEX, Foster City, CA) fueron spray 2.3kV tensión, temperatura de la fuente de iones de 150 ° C, GS1 de 12 años, y el gas cortina 15. </li><li> La muestra es analizada por SRM / MRM mediante la inyección de 10 l de la muestra. Áreas de los picos están integrados por péptidos ligeros y pesados, utilizando el programa de Skyline. 14 Calcular la relación del área de pico (PAR) del péptido no marcado / etiquetado de cada muestra con la transición más abundante que está libre de ruido de fondo, en este caso, la transición y5 (péptido luz 476,3&gt; 579,3, pesado estándar péptido 481,3&gt; 589,3). </li></ol> 4. Los resultados representativos: Medida ratios área del pico (en relación con la luz péptido endógeno de pinchos de péptidos marcados con isótopos pesados) proporcionan una medida cuantitativa del péptido objetivo. La figura 3 muestra un cromatograma de ejemplo de péptidos ligeros y pesados ​​en una muestra SISCAPA enriquecido. Tenga en cuenta que los péptidos ligeros y pesados ​​eluyen al mismo tiempo y múltiples transiciones pueden ser monitoreados para cada péptido para confirmar la identidad. <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="Figura 1"/> Figura 1. Un esquema resumen del proceso de SISCAPA. Una mezcla compleja de proteínas se digieren en péptidos. Objetivo analitos péptido (analito endógeno y pinchos de isótopos estables marcado patrón interno) se enriquecen con anticuerpos anti-péptido inmovilizado en la proteína G-revestido partículas magnéticas. Tras el aislamiento, los péptidos específicos son eluidos de las partículas magnéticas y analizados por espectrometría de masas para la cuantificación en relación con el patrón interno. <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="Figura 2"/> Figura 2. MS / MS espectro de la GDSLAYGLR péptido que muestra la selección de tres fragmentos de SRM / MRM transiciones. <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="Figura 3"/> Figura 3. Cromatogramas ejemplo que muestra el perfil de un pico de analito péptido (rojo) y el pesado de isótopos estables marcado patrón interno (azul). Cromatogramas de la transición y5 del péptido de luz (476,3&gt; 579,3) y el isótopo pesado estable etiqueta estándar (481,3&gt; 589,3) están representados a través del tiempo, ya que eluyen de la sistema de cromatografía.

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No hay conflictos de interés declarado.

El paso más crítico en el protocolo como se describe es asegurar las cuentas siguen siendo bien mezclada durante el período de incubación. Permitiendo que los gránulos se depositan en el fondo del pozo / vial se traducirá en una mayor variabilidad. También es importante para hacer girar hacia abajo el líquido que puede permanecer en la parte superior del pozo / vial tras el período de incubación. Digestión de la tripsina reproducibles también es fundamental. El procedimiento para la digestión descrito aquí se ha utilizado ampliamente en nuestro laboratorio en colaboración con SISCAPA, sin embargo, es probable que los métodos alternos de la digestión puede ser optimizado para un determinado conjunto de proteínas de la meta 15 de elución de los anticuerpos libres no interferir con la detección de la. péptido, probablemente debido a que el anticuerpo se excluye de la columna o eluye más tarde de los péptidos, pero el anticuerpo puede ser reticulado a las perlas de proteína G antes de enriquecimiento de afinidad en los experimentos de gran tamaño o si anticuerpo libre se convierte en un problema. También hemos encontrado que es útil para colocar un imán debajo de la placa de muestra en el muestreador automático, así como el lavado de la columna de la trampa en la dirección opuesta del flujo (en comparación con la carga) para eliminar cualquier residuo granos o partículas. Además de las bolas magnéticas enfoque descrito, la técnica también puede ser adaptado a un formato de columna (es decir, la cromatografía de afinidad). 12, 16 Una vez que el usuario se sienta cómodo con el protocolo general, la técnica también es susceptible de varias modificaciones que mejoran el ensayo general. 11 En primer lugar, es posible analizar múltiples analitos en un único ensayo por la combinación de anticuerpos en la etapa de enriquecimiento (multiplexación por ejemplo). El espectrómetro de masas es capaz de analizar simultáneamente un gran número de analitos. En segundo lugar, aumentar el volumen de la muestra original mejora la sensibilidad de la prueba.

<table border="1"><tbody><tr><th> Nombre del material </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> 1000ul placas de pozos profundos de 96 pozos </td><td> Eppendorf </td><td> 951032786 </td></tr><tr><td> Oasis HLB 1 cc cartucho de la placa, Sep-pak, 40 mg de placa de 96 pocillos </td><td> Aguas </td><td> 186003966 </td></tr><tr><td> Kingfisher placa de 96 pocillos </td><td> Thermo Fisher Scientific </td><td> 97002540 </td></tr><tr><td> Claro de 96, blanco placa de pared </td><td> Bio-Rad </td><td> HSP9601 </td></tr><tr><td> Papel de la cubierta de placa de 96 pocillos </td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> Axymat alfombra de sellado de placa de 96 pocillos </td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> X perforar la película </td><td> Sigma-Aldrich </td><td> Z722502 </td></tr><tr><td> Kingfisher magnética procesador de grano con la cabeza imán PCR </td><td> Thermo Fisher Scientific </td><td> HSP 9601 </td></tr></tbody></table> Tabla 1: Materiales <table border="1"><tbody><tr><th> Nombre del material </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Urea </td><td> Sigma Aldrich </td><td> U6031 </td><td></td></tr><tr><td> Trizma base de </td><td> Sigma Aldrich </td><td> T1503 </td><td></td></tr><tr><td> Ditiotreitol (DTT) </td><td> Atravesar </td><td> 20291 </td><td> &quot;No pesan ditiotreitol&quot; </td></tr><tr><td> Yodoacetamida (IAM) </td><td> Sigma Aldrich </td><td> I1149 </td><td></td></tr><tr><td> Tripsina Oro </td><td> Promega </td><td> V5280 </td><td></td></tr><tr><td> Proteína G magnética cuentas, 2.8um </td><td> Invitrogen </td><td> 10004D </td><td></td></tr><tr><td> Tampón fosfato salino (PBS) </td><td> Thermo Fisher Scientific </td><td> L5401 </td><td></td></tr><tr><td> CHAPS </td><td> Thermo Fisher Scientific </td><td> 28300 </td><td></td></tr><tr><td> Ácido fórmico </td><td> EMD </td><td> 11670 </td><td></td></tr><tr><td> Acetonitrilo </td><td> Thermo Fisher Scientific </td><td> A998-1 </td><td></td></tr><tr><td> El ácido acético </td><td> Sigma Aldrich </td><td> 242853 </td><td></td></tr></tbody></table> Tabla 2: Reactivos <table border="1"><tbody><tr><th> Solución </th><th> Comentarios (opcional) </th></tr><tr><td> 100 mM Tris pH 8 </td><td></td></tr><tr><td> 9 M urea / 30 mM de TDT en 100 mM Tris (pH 8) </td><td> debe prepararse cada vez </td></tr><tr><td> 500 mM IAM en 100 mM Tris (pH 8) </td><td> debe prepararse cada vez </td></tr><tr><td> Tripsina 1mg/ml en 100 mM Tris pH 8 </td><td></td></tr><tr><td> Inhibidor de tripsina de 1mg/ml en 100 mM Tris pH 8 </td><td></td></tr><tr><td> De isótopos estables estándar mastermix </td><td></td></tr><tr><td> Anticuerpos anti-péptido mastermix </td><td></td></tr></tbody></table> Tabla 3: Soluciones de estar preparados

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

Hay una gran necesidad de ensayos cuantitativos en la medición de las proteínas. Inmunoensayos tradicional bocadillo, en gran medida considera el estándar de oro en la cuantificación, se asocian con un alto costo, larga duración, y están llenos de inconvenientes (por ejemplo, anticuerpos heterófilos, la interferencia de autoanticuerpos, &#39;gancho-efecto &quot;). 1 Una técnica alternativa es el enriquecimiento de afinidad de péptidos acoplada a espectrometría de masas cuantitativa, comúnmente conocida como SISCAPA (Normas de isótopos estables y captura de anticuerpos anti-péptido). 2 En esta técnica, el enriquecimiento de afinidad de los péptidos con una dilución de isótopos estables y de detección por determinados / espectrometría de masa de reacción múltiple de control ( SRM / MRM-MS) proporciona una medición cuantitativa de los péptidos como sustitutos de sus respectivas proteínas. SRM / MRM-MS está bien establecida para la cuantificación exacta de las moléculas pequeñas 3, 4 y más recientemente ha sido adaptado para medir las concentraciones de proteínas en el plasma y lisados ​​celulares. 5.7 Para lograr la cuantificación de las proteínas, las moléculas de mayor tamaño son digeridos a péptidos componente usando una enzima como la tripsina. Uno o más péptidos seleccionados cuya secuencia es única a la proteína diana en la especie (es decir, &quot;proteotypic&quot; péptidos) son continuación de la muestra enriquecida con anticuerpos anti-péptido y se mide como sustitutos estequiométrica cuantitativa de la concentración de proteínas en la muestra. Por lo tanto, junto a la dilución de isótopos estables (SID) métodos (es decir, una enriquecida en isótopos estables etiquetados péptido estándar), SRM / MRM se puede utilizar para medir las concentraciones de péptidos proteotypic como sustitutos para la cuantificación de proteínas en matrices biológicas complejas. Los ensayos tienen varias ventajas en comparación con los inmunoensayos tradicionales. Los reactivos son relativamente menos caros para generar, la especificidad para el analito es excelente, los ensayos pueden ser altamente multiplexado, el enriquecimiento se puede realizar a partir de plasma puro (no requiere el agotamiento), y la técnica se presta a una amplia variedad de proteínas o modificaciones de interés. 8-13 En este video se demuestra el protocolo básico adaptado a una plataforma de cuentas magnéticas.

Watch this Scientific Journal Video about Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry at JoVE.com

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Normas de isótopos estables y de captura de anticuerpos anti-péptido (SISCAPA) enriquecimiento de las parejas de afinidad de los péptidos con una dilución de isótopos estables de espectrometría de masas (MRM-MS) para proporcionar una medición cuantitativa de los péptidos como sustitutos de sus respectivas proteínas. A continuación se describe el protocolo de uso de partículas magnéticas en un formato parcialmente automatizado.

La cuantificación de proteínas mediante el enriquecimiento de inmunoafinidad péptido acoplado a espectrometría de masas

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

quantification-proteins-using-peptide-immunoaffinity-enrichment

Research

JoVE Journal

Biology

22.7K vistas.  Fred Hutchinson Cancer Research Center - FHCRC. Normas de isótopos estables y de captura de anticuerpos anti-péptido (SISCAPA) enriquecimiento de las parejas de afinidad de los péptidos con una dilución de isótopos estables de espectrometría de masas (MRM-MS) para proporcionar una medición cuantitativa de los péptidos como sustitutos de sus respectivas proteínas. A continuación se describe el protocolo de uso de partículas magnéticas en un formato parcialmente automatizado.

Video: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

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

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Una evaluación cuantitativa de la Lipidome levadura utilizando la espectrometría de masas de ionización por electrospray

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

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 ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

En la cuantificación in vivo de interacciones de proteínas G del receptor acoplado con espectralmente resuelve de dos fotones de Microscopía

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Perfiles de metiltransferasas y Otros S-Adenosil- L-Homocisteína proteínas que se unen mediante espectrometría de masas de captura compuesto (MCP)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificación de los complejos de proteínas en<em&gt; Escherichia coli</em&gt; La purificación mediante afinidad del péptido secuencial en combinación con espectrometría de masas tándem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Captura compuesto Espectrometría de Masas - Una herramienta de gran alcance para identificar nuevos c-di-GMP efectoras Proteínas

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

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 ...