Os autores são gratos pelo apoio financeiro deste trabalho pela National Science Foundation EUA sob Grant No. 0719232, pelo Departamento de Energia dos EUA (DEFG02-01ER15129), e pela Protea Biosciences, Inc. (Morgantown, WV). Os autores também gostariam de agradecer a Jessica A. Stolee por sua ajuda durante a filmagem do protocolo.

O protocolo a seguir descreve as principais etapas da ionização electrospray a laser de ablação (LAESI) experiência e oferece exemplos representativos de lateral e imagens tridimensionais (3D) para animais e amostras de tecidos de plantas. Detalhes adicionais experimental e técnicas podem ser obtidas em outro lugar. 1-6 1. Preparação de tecido e de montagem <ol><li> Se o corte é necessário, use um cryomicrotome para a secção de tecido em 1-10 mM fatias grossas de -10 a -20 ° C a menos que seja recomendado para um tipo de tecido particular. </li><li> Montar seções em uma superfície plana (por exemplo, quimicamente lâmina de vidro previamente limpo) diretamente sem modificadores químicos. Para os tecidos seccionados, descongele-mount as seções e segura o porta-amostras para um estágio de resfriamento Peltier, imediatamente após a descongelação de montagem para manter os tecidos congelados em todos os momentos durante a análise. Este passo é necessário para minimizar / impedir a migração molecular na seção. </li><li> Se necessário, use um dissipador de calor equipados com um ventilador de baixa potência para facilitar a remoção de calor do Peltier palco para manter o tecido congelado. </li><li> Em um ambiente úmido durante um período prolongado de tempo (1-2 horas), para inspecionar a condensação de água ou gelo sobre a superfície do tecido. Condensação de água no tecido negativamente afeta o desempenho de imagem em experimentos LAESI 1. </li><li> Se necessário, use um desumidificador sala ou colocar a amostra resfriada em uma câmara ambiental preenchido com um gás inerte (por exemplo, o gás nitrogênio seco) para evitar a condensação 1. </li></ol> 2. Otimização da Fonte Ion LAESI A fonte de íons LAESI consiste de um laser infravermelho médio, uma série de elementos de óptica para direção de luz e foco, assim como porta-amostras adicionais, componentes de refrigeração, estágios de tradução, e um sistema de entrega de solvente. Figura 1 mostra o arranjo típico destes elementos com relação à entrada da fonte de íons atmosférica pressão de um espectrômetro de massa. <ol><li> Como ilustrado na figura 1, a posição da amostra 15-20 milímetros abaixo do orifício do cone de amostragem espectrômetro de massa (d OR-FP). </li><li> Operar um laser meados de IR em 2,94 mm e comprimento de onda 10 Hz taxa de repetição. Atenuar a saída do laser para ~ 100 μJ energia / pulso. </li><li> Use uma combinação de espelhos de ouro e uma lente com foco transparente no comprimento de onda do laser (por exemplo, um plano-convexa CaF 2 ou ZnSe lente) para acoplar a energia pulso de laser para a amostra a incidência normal (ângulo de incidência ver direito com respeito à superfície da amostra na Figura 1). </li><li> Posição do feixe de infravermelho médio eixo 5-8 mm na frente do orifício do cone de amostragem espectrômetro de massa. </li><li> Ajustar a posição da lente com foco ea energia de pulso do feixe de laser para alcançar a remoção de tecido no ponto focal. As dimensões do volume ablated determinar o pixel (ou voxel em imagens tridimensionais) de tamanho para a aplicação de imagem. </li><li> Posição de um emissor nanospray em linha com o eixo de entrada do espectrômetro de massa e em uma distância da ponta de orifício para emissor de ~ 10 mm (ver Figura 1). </li><li> Para o electrospray, prepare solução de metanol 50% com 0,1% de ácido acético ou acetato de aditivo de amônio 0,1% para o modo de íon positivo ou negativo, respectivamente. Dependendo da amostra, outros solventes orgânicos, como acetonitrila, isopropanol, etc, pode substituir o metanol em concentrações apropriadas para a tarefa analítica. A estabilidade do electrospray é crucial para a imagem de sucesso. Dependendo da selecção do solvente, a taxa de fluxo ea tensão pulverização precisa ser ajustada para atingir pulverização estável. </li><li> Para reativa LAESI 6 em aplicações de imagem, a solução electrosprayed pode conter reagentes. </li><li> Use uma bomba de seringa para entregar a solução electrospray através do emissor electrospray a uma vazão de ~ 300 nL / min. </li><li> Se o orifício espectrômetro de massa é mantida a uma baixa tensão (&lt;500 V medidos contra o chão), gerar electrospray através da aplicação de alta tensão diretamente para o emissor electrospray (eg, 3000 V) ou através de uma união metal. Caso contrário, terra diretamente emissor electrospray ou através da união de metal para estabelecer electrospray. </li><li> Operar a fonte electrospray no cone jet-pulverização modo de geração de íon mais eficiente por LAESI. Para o efeito de variáveis ​​operacionais sobre os modos de pulverização e seus efeitos sobre os espectros de massa, ver discussões em outros lugares. 5,7,8 </li><li> Ajustar cuidadosamente as distâncias relativas da configuração LAESI para otimizar o rendimento LAESI ion, mantendo o feixe de laser, o emissor, e os eixos orifício no mesmo plano. Por favor, encontrar instruções detalhadas em outros lugares. 5 </li><li> Com um microscópio óptico, determine as dimensões laterais da cratera ablação na amostra. </li><li> Para tridimensional experimentos com imagens LAESI, a realização da ablação com pulsos individuais e determinar a profundidade de um voxel usando, por exemplo, o modo de z-stack em microscopia óptica. 3 </li></ol> 3. Imagem Molecular e Análise de Dados No experimento de imagem, a amostra de tecido é movido no plano focal do laser em X e Y direções com tamanhos de passo maior do que ou iguais às dimensões do local de ablação. A resolução espacial é limitada pela focalização do feixe de laser incidente. <ol><li> Selecione a área de interesse na superfície da amostra e obter o (X, Y) coordenadas dos limites correspondentes. </li><li> Escolheu um algoritmo gridding (eg, adaptação de grade, imagem região selecionada, grade retangular, espiral, Z digitalização, etc) com o qual raster a superfície da amostra selecionada, com tempo de permanência em cada pixel sobre a área a ser trabalhada. </li><li> Use um estágio tradução de três eixos e software que é capaz de rastering a amostra de acordo com a grelha pré-determinado. </li><li> Calcule o tempo total necessário para geração de imagens. </li><li> Desativar os dados limite de tempo de aquisição do espectrômetro de massa. Se isso não for possível, definir o prazo de aquisição de dados com o valor calculado imagem tempo. </li><li> Iniciar a fonte de laser infravermelho médio uma taxa de repetição adequada para produzir a relação sinal-ruído suficiente no espectro de massa dentro do tempo de permanência em cada pixel para realizar um experimento de imagem LAESI lateral. Para a imagem latente molecular 3D, use uma taxa de aquisição de espectro maior do que a taxa de repetição do laser fonte com sucesso em massa analisar os íons gerados dentro de um único pulso de laser. Aguarde o sinal de partida para iniciar a seqüência ablação. </li><li> Ligue a fonte de electrospray. Certifique-se que não há solução suficiente durante todo o tempo necessário para geração de imagens. </li><li> Simultaneamente iniciar a aquisição de espectros de massa, a ablação a laser meados de IR, ea varredura de superfície. </li><li> Quando a execução de imagens terminar, parar a varredura de superfície, o lasing meados de IR, ea aquisição de dados. </li><li> Desabilitar a fonte laser. </li><li> Desligue a alta tensão. </li><li> Desligue a bomba de seringa. </li><li> Definir o espectrômetro de massa para o modo STANDBY. </li><li> Desligue o sistema eletrônico de refrigeração Peltier. </li><li> Fechar o fluxo de gás inerte, se usado. </li><li> Use um software para correlacionar as coordenadas absolutas dos pixels na imagem lateral ou os voxels na análise 3D com os espectros correspondentes. </li><li> Plotar a intensidade de sinal de íons para selecionar o valor m / z contra o coordenadas absolutas de análise para obter lateral e imagens 3D molecular. </li></ol> 4. Resultados representante A Figura 2 apresenta resultados representativos para alguns tipos de tecidos importantes e modalidades de imagem. Um painel retrata um caso para uma seção de tecido animal que tenha sido congelada durante o experimento para evitar a desidratação. 1 Além disso, a amostra foi localizado em um ambiente seco gás nitrogênio para evitar vapores de água ambientes de condensação na superfície da amostra. Uma seção de 100 m de espessura coronal de um cérebro de rato (Rattus norvegicus) foi fotografada com LAESI lateralmente. As regiões anatômicas do cérebro (veja a imagem óptica no painel A) apresentam boa correlação com a imagem molecular obtido para o PC plasmalógenos (O-33: 3) e / ou PE (O-36: 3) com m / z 728,559. Painel B mostra a imagem LAESI 3D de uma planta Zebra (Aphelandra squarrosa) tecido foliar. Porque as folhas possuem um mecanismo natural de defesa contra a desidratação, a amostra poderia ser interrogado no ambiente ambiente. 3 As imagens 3D obtidas molecular revelou uma variedade de padrões de distribuição de metabólitos primários e secundários. Entre outros, acacetin com m / z 285,076 foi detectada na maior contagem de íons nos setores amarelo da segunda e terceira camadas de cima com uma distribuição homogênea nos outros. Esta distribuição concordou com o padrão da variegação visto na imagem óptica. <img src="/files/ftp_upload/2097/2097fig1.jpg" alt="Figura 1" /> Figura 1. Esquemática do sistema LAESI (ES, electrospray ponta emissor; OR, orifício do cone espectrômetro de massa de amostragem; FL, com foco da lente; FP, ponto focal; P, Peltier estágio de resfriamento; HS, dissipador de calor). Uma parte do material particulado expelido durante meados de IR de ablação (pontos vermelhos) funde com o electrospray para produzir gotas cobrado semeado com íons e moléculas da amostra (pontos verdes). Os íons liberados essas gotículas são analisados ​​e registrados pelo espectrômetro de massa. <img src="/files/ftp_upload/2097/2097fig2.jpg" alt="Figura 2" /> Figura 2. Resultados representativos para Imagi lateral e 3D. ng com LAESI espectrometria de massa (A) O painel superior mostra a imagem óptica de um cérebro de rato (Rattus norvegicus) seção coronal ea imagem molecular obtido para o PC plasmalógenos (O-33: 3) e / ou PE (O-36 : 3) com m / z 728,559. A barra de escala em branco corresponde a 1 mm. Adaptado com permissão de (referência 1). Copyright 2010 American Chemical Society. (B) O painel inferior mostra a imagem 3D de uma folha de uma planta variegada Zebra (Aphelandra squarrosa). Acacetin com m / z 285,076 foi detectada na maior contagem de íons nos setores amarelo da segunda e terceira camadas de cima com uma distribuição homogênea nos outros. Reproduzido com permissão do (Referência 3). Copyright 2009 da Sociedade Americana de Química.

<ol>
	<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> Anal Chem. 82, 982-988 (2010).</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> Anal Chem. 79, 8098-8106 (2007).</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> Anal Chem. 81, 6668-6675 (2009).</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> Anal Chem. 80, 4575-4582 (2008).</li><li>Nemes, P., Vertes, A., Nemes, S. S., Rubakhin, J. V. <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+molecular+imaging+mass+spectrometry.+Mass+Spectrometry+Imaging.">Laser ablation electrospray ionization for atmospheric-pressure molecular imaging mass spectrometry. Mass Spectrometry Imaging.</a> Methods in Molecular Biology. , (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. 135, 751-758 (2010).</li><li>Nemes, P., Marginean, I., 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=Spraying+mode+effect+on+droplet+formation+and+ion+chemistry+in+electrosprays.">Spraying mode effect on droplet formation and ion chemistry in electrosprays.</a> Anal Chem. 79, 3105-3116 (2007).</li><li>Nemes, P., Goyal, 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=Conformational+and+noncovalent+complexation+changes+in+proteins+during+electrospray+ionization.">Conformational and noncovalent complexation changes in proteins during electrospray ionization.</a> Anal Chem. 80, 387-395 (2008).</li><li>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=Molecular+imaging+by+Mid-IR+laser+ablation+mass+spectrometry.">Molecular imaging by Mid-IR laser ablation mass spectrometry.</a> Appl Phys A-Mater Sci Process. 93, 885-891 (2008).</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> Anal Chem. 81, 8265-8271 (2009).</li><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></ol>

Não há conflitos de interesse declarados.

Diferentes tipos de tecidos apresentam diferentes teores de água e resistência à tração, que, por sua vez, pode afetar as características de ablação das amostras. 9 Para atenuar esses efeitos, é desejável que os protocolos a fluência do laser, a manipulação da amostra, ea análise deve ser revisto quando se muda entre principais tipos de tecido. Única célula ou para investigações de alta resolução, a luz infravermelho médio pode ser acoplado em uma fibra óptica afiada em vez de uma lente de foco. 10 Ao posicionar a ponta de fibra nas proximidades das células selecionadas em um tecido, a análise LAESI pode ser executada em um nível de uma única célula. Como um rótulo sem fonte de íons para a espectrometria de massa de ionização do ambiente, 11 LAESI tem mostrado grande potencial para a investigação de processos bioquímicos nos tecidos. Com os benefícios adicionais de análise direta, lateral e de imagem 3D, LAESI é uma ferramenta para criação de perfis emergentes bioanalíticos, bem como aplicações de imagem.

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<td>Mass spectrometer</td>
<td>&nbsp;</td>
<td>Waters Co. (Milford, MA)</td>
<td>Q-TOF Premier</td>
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<td>Mid-IR laser</td>
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<td>Opotek Inc. (Carlsbad, CA)</td>
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atmospheric-pressure molecular imaging of biological tissues and biofilms by laesi mass spectrometry

Métodos de ionização ambiente em espectrometria de massa permitem investigações analíticas para ser realizada diretamente em um tecido ou biofilme sob nativo como condições experimentais. Laser de ionização electrospray ablação (LAESI) é um tal desenvolvimento e é particularmente adequado para a investigação de água contendo espécimes. LAESI utiliza um feixe de laser infravermelho médio (2,94 de comprimento de onda mm) para excitar as moléculas de água da amostra. Quando o limiar de influência da ablação é excedido, o material da amostra é expelido na forma de partículas e estes projéteis viajar para dezenas de milímetros acima da superfície da amostra. Em LAESI, esta pluma de ablação é interceptada por gotas altamente carregadas para capturar uma fração do material de amostra ejetado e converter seus constituintes químicos em fase gasosa íons. Um espectrômetro de massa equipados com uma interface de origem atmosférica pressão íon é empregado para analisar e registrar a composição dos íons liberados oriundos da área sondada (pixel) da amostra. A interrogação sistemática sobre uma matriz de pixels abre caminho para a imagem latente molecular no modo de análise de microssonda. Um aspecto único do LAESI imagem espectrometria de massa é de perfis de profundidade que, em combinação com a imagem lateral, permite imagens tridimensionais (3D) molecular. Com as atuais resoluções lateral e profundidade de ~ 100 mm e ~ 40 mM, respectivamente, LAESI imagem espectrometria de massa ajuda a explorar a estrutura molecular de tecidos biológicos. Aqui, revisamos os principais elementos de um sistema LAESI e fornecer orientações para uma experiência bem sucedida de imagem.

Watch this Scientific Journal Video about Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry at JoVE.com

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

Laser de ionização electrospray ablação (LAESI) é uma fonte de íons na atmosfera de pressão para a espectrometria de massa. No modo de imagem, um sondas de laser infravermelho médio das distribuições de moléculas através de uma secção de tecido ou de um biofilme. Esta técnica apresenta uma nova abordagem para diversos estudos bioanalíticos realizadas sob condições experimentais nativa.

Pressão atmosférica Imagem Molecular de tecidos biológicos e Biofilmes por LAESI Espectrometria de Massa

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

atmospheric-pressure-molecular-imaging-biological-tissues-biofilms

Nanyang Technological University

Research

JoVE Journal

Biology

14.1K visualizações.  George Washington University. Laser de ionização electrospray ablação (LAESI) é uma fonte de íons na atmosfera de pressão para a espectrometria de massa. No modo de imagem, um sondas de laser infravermelho médio das distribuições de moléculas através de uma secção de tecido ou de um biofilme. Esta técnica apresenta uma nova abordagem para diversos estudos bioanalíticos realizadas sob condições experimentais nativa.

Vídeo: Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI 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 preparação da amostra: o método de Camada Ultra Thin

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

Biologia

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.

Analisar grandes complexos de proteínas por espectrometria de massa estruturais

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.

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

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

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.

Análise direta de células individuais por Espectrometria de Massa à pressão 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.

Profiling Proteome Redox tiol Usando Isótopos Espectrometria de Massa Tagging

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Tandem congelamento de pressão alta e rápida substituição em congelamento dos tecidos vegetais por Microscopia Eletrônica de Transmissão

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

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.

Enriquecimento de Proteínas da Matriz Extracelular a partir de tecidos e digestão em péptidos para Análise de Espectrometria de Massa

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.

Aplicações da sonda Individual: Imagem Espectrometria de Massa e Análise única célula sob condições ambientais

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

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, medial-Golgi, trans-Golgi and trans-Golgi network. Cellular functions of the Golgi are determined by the characteristic distribution of its resident proteins. The spatial resolution of conventional light microscopy is too low to resolve sub-Golgi structure or cisternae. Thus, the immuno-gold electron microscopy is a method of choice to localize a protein at the sub-Golgi level. However, the technique and instrument are beyond the capability of most cell biology labs. We describe here our recently developed super-resolution method called Golgi protein localization by imaging centers of mass (GLIM) to systematically and quantitatively localize a Golgi protein. GLIM is based on standard fluorescence labeling protocols and conventional wide-field or confocal microscopes. It involves the calibration of chromatic-shift aberration of the microscopic system, the image acquisition and the post-acquisition analysis. The sub-Golgi localization of a test protein is quantitatively expressed as the localization quotient. There are four main advantages of GLIM; it is rapid, based on conventional methods and tools, the localization result is quantitative, and it affords ~ 30 nm practical resolution along the Golgi axis. Here we describe the detailed protocol of GLIM to localize a test Golgi protein.

The precise localization of Golgi residents is essential for understanding the cellular functions of the Golgi. However, conventional optical microscopy is unable to resolve the sub-Golgi structure. Here we describe the protocol for a conventional microscopy based super-resolution method to quantitatively determine the sub-Golgi localization of a protein.

Localização de quantitativa de uma proteína de Golgi por imagem sua massa centro de fluorescência

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...