Os autores agradecem o apoio financeiro das seguintes instituições: a National Science Foundation (Grant 0719232), o Departamento de Energia (Grant DEFG02-01ER15129), o WM Keck Foundation (Grant 041.904), ea George Washington University Fundo Enhancement Research. Os autores são gratos pelas fibras ópticas GeO2 baseado generosamente fornecida por sistemas de fibra de infravermelhos (Silver Spring, MD), bem como para a discussão inicial sobre o protocolo sobre gravação de fibra por Mark E. Reeves e Joan A. Hoffmann da George Washington University. Os autores também gostariam de agradecer a Jessica A. Stolee por sua ajuda durante a filmagem do protocolo.

1. Componentes ópticos <ol><li> Ablação a laser para o laser de ionização electrospray ablação (LAESI) espectrometria de massa (MS) é produzido por um laser de 5 ns pulso em comprimento de onda 2,94 mM. Um oscilador paramétrico óptico sintonizável impulsionada por um laser Nd: YAG (Opolette 100, Opotek Inc., Carlsbad, CA) é usado para ablação neste estudo. A taxa de repetição é selecionado entre 5 e 100 Hz. Este é um laser classe IV que após a exposição direta pode causar danos permanentes graves nos olhos ou da pele. A reflexão difusa do feixe de laser também podem ser perigosos para a pele ou olhos. Usar um óculos de laser adequados de protecção. </li><li> A fibra óptica feitos de germânio de vidro baseado no dióxido de infravermelho foi obtido a partir Fiber Systems, Inc. (Silver Spring, MD). Se houver Hytrel e poliimida revestimentos em fibra siga as instruções abaixo para removê-los a partir das extremidades. Heat 1-metil-2-pirrolidinona a 130-150 ° C em uma capela. Mergulhe as extremidades da fibra que você quer retirar este líquido em cerca de 1 min ou até que o revestimento começa a amolecer e descolar. Mergulhe o revestimento amolecida em metanol ou isopropanol por um minuto, e limpe qualquer revestimento restantes com um guardanapo que não solte fiapos. Após a remoção do revestimento, cleave ambas as extremidades da fibra com uma lâmina de safira (Kitco Fibra Óptica, Virginia Beach, VA) marcando e delicadamente tirando-los. </li><li> Para alcançar a nitidez desejada, quimicamente uma extremidade do etch ponta da fibra em 1% (v / v) de ácido nítrico (grau de reagente) solução. Verticalmente imergindo a extremidade da fibra selecionados para a solução de ácido a uma profundidade de 300 a 500 mm após o contacto inicial. Após aproximadamente 15 minutos de gravação, a dica vai separar-se espontaneamente a partir da superfície do ácido. Isso resulta em uma ponta da fibra afiada com ~ 15 mM raio de curvatura. Enxágüe com água deionizada para remover qualquer resíduo do ácido. </li><li> Montagem final gravado da fibra em um micromanipulador (MN-151, Narishige, Tóquio, Japão) e trazê-lo à proximidade (~ 20 mm) da superfície da célula para deposição de energia eficiente. </li><li> Segure a extremidade não-gravado da fibra óptica por um bare fibra chuck (BFC300, Siskiyou Corporation, Grants Pass, OR). Para o alinhamento óptico, o chuck fibra é montado em um tradutor cinco eixos (BFT-5, Siskiyou Corporation, Grants Pass, OR). A energia do laser (0,5 a 1 mJ) é acoplado na fibra por foco o feixe por um fluoreto de cálcio plano-convexo ou lente anti-reflexo ZnSe revestido (Infrared Produtos Ópticos, Farmingdale, NY) para o seu fim não-gravadas. Espelhos de ouro protegidas (PF10-03-M01, Thorlabs, Newton, NJ) são utilizados para orientar o feixe de laser meados de IR sobre a lente de foco. </li></ol> 2. Setup electrospray <ol><li> O sistema de electrospray pode ser construído usando os seguintes componentes: a união do metal com ponteira perfluoroelastômero condutores, conexões, tubos de manga, porta agulha (por exemplo, U-435, M215, F-331Nx, F-242x, 9013, respectivamente IDEX Health &amp; Sciences, Oak Harbor, WA), bem como tubos de sílica fundida (por exemplo, CT360-100-50-5, Nova Objetivo Inc., Woburn, MA). O emissor recomendado é feito de aço inoxidável e tem uma ponta cônica com um ID de 50 mm (por exemplo, MT320-50-5-5, Nova Objetivo Inc., Woburn, MA). </li><li> Prepare a solução de metanol a 50% com 0,1% de ácido acético (v / v) para o electrospray. A solução é bombeada a uma taxa de 200 a 300 nl / min por uma bomba de seringa (Harvard Apparatus, Holliston, MA), utilizando, por exemplo, um. Seringa 500 mL (81.222, empresa Hamilton, Reno, NV) </li><li> Aplicar alta voltagem entre 2.800 e 3.000 V para a união de metal por uma fonte de alimentação regulada (PS350, Stanford Research Systems Inc, Sunnyvale, CA) para gerar um electrospray estável. Certifique-se de todas as conexões elétricas estão seguras e blindado com a blindagem aterrada. Contato direto com expostos de alta tensão pode resultar em choque elétrico. </li></ol> 3. Manuseamento da amostra <ol><li> Obter o tecido vivo ou amostra de células a partir de uma fonte adequada e mantê-los como recomendado antes da análise. As lâmpadas Allium cepa para esta demonstração foram compradas de uma loja local. Cortar uma lâmpada longitudinalmente por um bisturi cirúrgico. Um segmento de poucos centímetros de uma escala foi seccionada em uma tira. Uma monocamada intacta do tecido interior da epiderme é removida. </li><li> A superfície molhada da epiderme é usado para montar o tecido sobre uma lâmina pré-limpeza de vidro de microscópio. Segure a lâmina por um detentor de propósito geral da placa (FP01, Thorlabs Inc., Newton, NJ) montada em um palco tradução de três eixos para o posicionamento. Adquirir os espectros de massa imediatamente após a montagem da amostra para evitar a degradação celular. </li></ol> 4. Sistema de Visualização <ol><li> Dois microscópios de longa distância são usados ​​para selecionar as células para análise e para manter a distância entre a ponta afiada de fibra ea superfície da célula. O último é montado sob 15 ° ângulo de -30 medido a partir da superfície. Este sistema consiste em along microscópio de vídeo à distância (7x zoom óptico de precisão, Edmund Optics, Barrington, NJ), com uma infinidade de 5x corrigido lente objetiva (M Apo Plano de 5x, Mitutoyo Co., Kanagawa, Japão) equipado com uma câmera CCD (Marlin F131, a Allied Vision Technologies , Stadtroda, Alemanha) para monitoramento e captura de imagem. </li><li> O microscópio segundo vídeo é montado em um ângulo reto com a superfície da amostra para visualizar as células a partir do topo. Este sistema fornece feedback visual para alinhar a ponta da fibra ao longo da célula selecionada para ablação e análise. Ele consiste de um zoom óptico de 7x de precisão (Edmund Optics, Barrington, NJ), equipada com uma infinidade 10x corrigido longa lente objetiva do trabalho à distância (M Plano de Apo 10x, Mitutoyo Co., Kanagawa, Japão) e uma câmera CCD. </li></ol> 5. Aquisição de espectros de massa <ol><li> Ligue o laser, o electrospray eo espectrômetro de massa, por exemplo, um sistema de tempo de vôo quadrupolo (Q-TOF Premier, Waters, Milford, MA). Otimizar a geometria da fonte de íons LAESI ajustando a posição do ponto de ablação e emissor electrospray em relação ao outro eo orifício do espectrômetro de massa para atingir intensidades máximas de íons. </li><li> Selecione a célula de interesse para análise usando o sistema de visualização top-view. Após a seleção, mova o estágio de amostra para levar a célula selecionada diretamente abaixo da ponta afiada da fibra óptica. Usando o microscópio de visão lateral reajustar a ponta-a-célula de distância da superfície para ~ 20 mM. </li><li> Pulsos de laser de fogo na célula selecionada através da ponta de fibra gravado. Este resultado na perfuração da parede celular e da ejeção do citoplasma na forma de partículas. Depois postionization pela electrospray, os íons produzidos são coletados pelo espectrômetro de massa e espectros de massa são registradas. </li><li> Após a aquisição de dados, colocar o laser, o electrospray eo espectrômetro de massa no modo de espera ou desligá-los. Manter o A. ablated cepa amostra de tecido para a observação opcional por microscopia de luz. </li></ol> 6. Resultados representante Ablação bem sucedida de uma única célula resultados na explosão da parede celular e à recolha de um espectro de massa LAESI. Uma experiência mal sucedida geralmente não resulta em ablação e / ou nenhum espectro de massa. Para fins de demonstração, apresentamos os resultados da análise LAESI-MS de uma célula simples incorporada epidérmica de uma A. bulbo cepa. Figura 2a mostra uma imagem de microscopia óptica após a representante de amostragem de uma célula. A parede celular é perfurado ea extensão da perfuração é limitada pelas fronteiras com as células vizinhas. As células adjacentes parecem estar intactos. Figura 2b mostra um espectro de massa representante LAESI produzido a partir da célula. Uma grande variedade de íons são detectados a partir do A. cepa da célula e com base em suas massas precisas, padrões de distribuição de isótopos, e, em alguns casos, tandem espectros de massa, são tentativamente atribuído aos metabólitos endógenos. Os íons detectados a partir de uma única célula foram semelhantes aos observados na análise do mesmo tecido por convencionais LAESI-MS de várias células 13. <img src="/files/ftp_upload/2144/2144fig1.jpg" alt="Figura 1" /> Figura 1. Esquemática de análise única célula por LAESI-MS. A meados de IR pulso de laser é acoplado em uma fibra óptica e entregue com a amostra colocada sobre lâmina de vidro. A ponta da fibra afiada focaliza a luz para dentro da célula-alvo e da energia depositada rajadas da célula. A pluma de ablação produzida (pontos vermelhos) é mesclada com uma pluma electrospray (pontos pretos) as gotas coalescem (pontos verdes), resultando em amostra de produção de íons específicos. Esses íons são analisados ​​e detectados pelo espectrômetro de massa (MS). Dois microscópios de longa distância de vídeo, Microscópio Microscópio 1 e 2, são utilizados para monitorar a distância entre a ponta da fibra e da superfície celular e selecionar uma célula para análise, respectivamente. <img src="/files/ftp_upload/2144/2144fig2a.jpg" alt="Figura 2a" /> Figura 2a marca. Ablação em uma única célula epidérmica de uma A. bulbo cepa. As células adjacentes não apresentam danos visíveis e, posteriormente, podem ser analisados ​​separadamente. <img src="/files/ftp_upload/2144/2144fig2b.jpg" alt="Figura 2b" /> Figura 2b Espectro de massa. Correspondentes à ablação de um único A. cepa de células epidérmicas mostra a presença de metabólitos primários e secundários.

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O conteúdo de água no citoplasma da célula e resistência à tração da parede celular ou membrana são fatores críticos que regem a ablação de células isoladas durante LAESI-MS análise. A influência do laser tem que ser ajustada para atingir os limiares de ablação dos tipos de células diferentes. A influência do laser entregues depende da energia do pulso de laser e na distância entre a ponta da fibra e da superfície da célula. Minimizar a perturbação de células vizinhas durante a análise única célula é crítica. Manter a energia do laser no limiar de ablação limita o efeito de análise sobre as células adjacentes. Uma das aplicações possíveis é usando células individuais como os pixels naturais volumétrica (voxels) para produtos químicos de imagem por MS. Em comparação com a coleta de dados em uma grade retangular artificial, muitas vezes, química imagem de uma célula do tecido de cada vez é mais provável que mantenham a organização espacial das interações bioquímicas entre as células.

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direct analysis of single cells by mass spectrometry at atmospheric pressure

Análise de produtos bioquímicos em células individuais é importante para a compreensão do metabolismo celular, ciclo celular, adaptação, estados de doença, etc Mesmo os tipos de células apresentam mesma composição bioquímica heterogêneos dependendo de suas condições fisiológicas e interações com o ambiente. Métodos convencionais de espectrometria de massa (MS) utilizados para a análise de biomoléculas em células individuais dependem de preparação da amostra extensa. Removendo as células do seu ambiente natural e processamento da amostra extensa pode levar a mudanças na composição celular. Métodos de ionização do ambiente permitem a análise de amostras em seu ambiente nativo e sem a preparação da amostra extensa. 1 As técnicas com base no infravermelho médio ablação a laser (meados de IR) de materiais biológicos em comprimento de onda 2,94 mM utilizar a excitação repentina de água que resulta em fase de explosão. duas técnicas de ionização ambiente baseado em meados de IR radiação laser, como laser de ionização electrospray ablação (LAESI) e pressão atmosférica infravermelho matrix-assisted laser de ionização de dessorção (AP-IR MALDI), têm demonstrado com sucesso a capacidade de analisar diretamente a água tecidos ricos e biofluidos à pressão atmosférica. 3-11 LAESI Em meados da década de IR pluma de ablação a laser que consiste principalmente de partículas neutras do aglutina amostra com gotas electrospray altamente carregada de produzir íons. Recentemente, em meados de IR ablação de células isoladas foi realizada através da apresentação da radiação meados de IR através de uma fibra gravado. A pluma gerada a partir deste ablação foi postionized por um electrospray permitindo a análise de metabólitos diversos em uma única célula por LAESI-MS. 12 Este artigo descreve o protocolo para a análise detalhada única célula usando LAESI-MS. O vídeo apresentado demonstra a análise de uma única célula epidérmica da pele de uma lâmpada Allium cepa. O esquema do sistema é mostrado na Figura 1. Um exemplo representativo de ablação única célula e espectrometria de massa LAESI da célula são fornecidos na Figura 2.

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

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Análise única célula é realizada por espectrometria de massa de células vegetais e animais à pressão atmosférica usando uma fibra óptica afiada para provar as células para o laser de ionização electrospray ablação (LAESI) espectrometria de massa.

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 ...

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Biology

15.9K visualizações.  George Washington University. Análise única célula é realizada por espectrometria de massa de células vegetais e animais à pressão atmosférica usando uma fibra óptica afiada para provar as células para o laser de ionização electrospray ablação (LAESI) espectrometria de massa.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

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

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

MALDI 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. ...

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

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

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

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 ...

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

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

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

Amida de hidrogênio / deutério Exchange &amp; MALDI-TOF Análise Espectrometria de Massa de PAK2 Ativação

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

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

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

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

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

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

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 ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

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

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

Análise de LINE-1 retrotransposição no Núcleo de nível único

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

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

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

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

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 ...

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

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

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

Preparação da amostra para análise de Proteomics baseados em massa-espectrometria de Microvessels Ocular

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