Este trabalho foi financiado pela Clínica NCI Tecnologia Proteômica Assessment Center (CPTAC) conceder (# U24 CA126476), bem como uma concessão da indústria do entretenimento Foundation (FEI) eo Fundo das Mulheres FEI Pesquisa do Câncer da Mama Consórcio Descoberta Cancer Biomarker, e generosas doações da Fundação Keck, a Fundação Canárias e dos Paul G. Allen Família Foundation.

Procedimento experimental: O ensaio requer peptídeos sintéticos e anticorpos anti-peptídeo. Peptídeos selecionados deve ser exclusivo para a proteína de interesse, conter entre 8 e 22 aminoácidos, e não tem conhecido modificações pós-translacionais. Resíduos de metionina são geralmente evitada e peptídeos contendo aminoácidos dibásicos (por exemplo, KK, KR, RR) são indesejáveis. Para esta técnica, é comum o uso de peptídeos isótopo estável rotulados como normas internas, incorporando pesado (13 C e 15 N) marcado aminoácidos no C-terminal do peptídeo (ou seja, K ou R rotulados). O protocolo a seguir descreve um teste desenvolvido para medir a GDSLAYGLR peptídeo da proteína Osteopontina mouse, utilizando anticorpos anti-peptídeo obtido a partir de Epitomics Inc. (Burlingame, CA) e peptídeos sintéticos da Nova Inglaterra Peptide (Gardner, MA). O protocolo consiste em três etapas principais (Figura 1): 1) Tripsina digestão das proteínas complexas, 2) Enriquecimento de peptídeos 3) Análise por espectrometria de massa. Será demonstrado em uma amostra de plasma humano enriquecida com a proteína Osteopontina mouse. 1. Tripsina digestão enzimática e limpeza <ol><li> Thaw 10 mL alíquota plasma puro em gelo molhado. </li><li> Determinar a concentração de proteínas totais por ensaio BCA e centrifugar a amostra para remover quaisquer sólidos em suspensão. </li><li> Pipeta alíquota de 10 L de seu tubo de armazenamento para uma placa Deepwell 1000 mL e cubra com pierce-able filme. </li><li> Adicionar 20 mL de 9M fresco uréia / 30mm ditiotreitol (DTT) (final de concentração 6M TDT uréia / 20mm) para cada amostra. </li><li> Incubar por 30 minutos a 37 ° C. </li><li> Adicionar 3 mL de iodoacetamida mM fresco 500 (IAM final de 50 mM) para cada amostra. </li><li> Incubar por 30 minutos no escuro à temperatura ambiente. </li><li> Adicionar 257 mL de 100 mM Tris (pH 8) (dilui uréia para 0,6 M ~). </li><li> Adicionar 10 mL de tripsina solução estoque (1 mg / mL; para 01:50 enzima: substrato). </li><li> Incubar 37 ° C durante a noite (12-16 horas). </li><li> Adicionar 3 mL de ácido fórmico pura (concentração final de 1%). </li><li> Adicionar padrão de isótopos estáveis ​​(padrões múltiplos são adicionados se realizar um ensaio multiplex, geralmente essa é cerca de 10 mL contendo 5-10 fmol de peptídeo isotopicamente marcado standard). </li><li> Lavar a placa do cartucho Oasis bem com 500 mL de 0,1% de ácido fórmico em acetonitrila 80%, descartando o escoamento. Repita 3 vezes. </li><li> Equilibrar a placa do cartucho, adicionando 500 mL de 0,1% de ácido fórmico em água, e descartar o escoamento. Repita isso 4 vezes. </li><li> Carregar amostras digerir a placa do cartucho e ajustar o vácuo de modo que o fluxo é muito lento. </li><li> Lavar com 500 mL de 0,1% de ácido fórmico em água, e descartar por escoamento. Repita 3 vezes. </li><li> Peptídeos eluir pela adição de 2 x 500 mL de 0,1% de ácido fórmico em acetonitrila 80% em 1000 prato fundo bem-mL (não descartam a flow-through). </li><li> Lyophilize (ou speedvac) o eluído até à dessecação. (Liofilização é o método preferido) </li><li> Reconstituir secas peptídeos pela adição de 50 mL PBS + 0,03% CHAPS. </li></ol> 2. Peptídeo de enriquecimento de imunoafinidade <ol><li> Transferir a amostra a norma Kingfisher 96 placas bem. </li><li> Adicionar um anticorpo mg e 1,5 mL da proteína G-esferas magnéticas revestidas por alvo (que é opcional para crosslink o anticorpo para as contas antes disso). Garantir contas são bem suspenso por agitação ou vórtex. </li><li> Cobrir a placa com papel alumínio. </li><li> Incubar overnight (12-16 horas) com suave caindo para garantir contas são suspensas. </li><li> Centrífuga placa a 32 xg por 5 segundos para remover qualquer líquido a partir da superfície da folha. </li><li> Remova a cobertura de alumínio. </li><li> Lavagem e eluição das contas pode ser feita manualmente ou de forma automatizada. Este procedimento descreve as etapas automatizadas em uma plataforma de Kingfisher. </li><li> Lave as contas duas vezes com 200 mL PBS + 0,03% CHAPS (1 minuto por lavagem). </li><li> Lave as contas uma vez com 200 mL de diluição de 1:10 de PBS + CHAPS 0,03% (1 minuto). </li><li> Os peptídeos são eluídos em 25 uL de 5% de ácido acético + CHAPS 0,03%. </li><li> Coloque a placa de eluição em um ímã e transferir o eluato para um prato de 96 poços, tendo o cuidado de não transferir as contas sobra. </li><li> Cobrir a placa com um tapete de vedação. </li><li> A placa contendo o eluato é entregue ao espectrômetro de massas triplo quadrapole para análise. </li></ol> 3. Análise por monitoramento de reações múltiplas - espectrometria de massa <ol><li> Transições para SRM / MRM análise podem ser selecionados e otimizados pela infusão de uma solução picomole 1 por microlitro do padrão de peptídeos em 30% de ácido fórmico acetonitrile/0.1% para o espectrômetro de massa de 0,5 mL / min. Uma vez de spray estável é obtido, recolher um espectro de MS / MS. </li><li> Transições são selecionados a partir do espectro MS / MS, identificando three íons fragmento abundantes em uma região do espectro com pouco ruído. Para multiplicar-íons carregados peptídeo, os fragmentos y-ion detectado em m / z&gt; precursor m / z são normalmente o melhor para o SRM / MRM desenvolvimento ensaio. Figura 2 mostra o espectro de MS / MS e íons de transição selecionados 476,3&gt; 508,3, 579,3 , 692,4 (transições para o pesado isótopo estável padrão não 481,3&gt; 518,3, 589,3, 702,4 mostrado) para a GDSLAYGLR peptídeo. </li><li> Dependendo da marca e modelo do espectrômetro de massa utilizada, existem vários parâmetros que podem ser otimizados para cada transição. Aqui, nós otimizamos a energia de colisão de cada transição selecionados por ramping a energia da colisão e monitoramento do nível de sinal. A energia de colisão de 25 foi usado para cada transição. </li><li> Resumidamente, uma configuração típica para SRM / MRM análise é a seguinte: Fase móvel (A) ácido fórmico 0,1%, (B) acetonitrila 90% / 0,1% de ácido fórmico, 0,3 x 5 mm coluna armadilha C18, 75 mM ID x 15 cm coluna analítica C18 (Reprosil-Pur AQ C18, 120 A poros °). O volume de injeção é de 10 mL da amostra e é carregado por 5 min a B 3% a uma vazão total de 3 mL / min e eluída por um gradiente linear 3-45% B no 300-400 nL / min em 10 min. Condições em um QTRAP 4000 (ABSCIEX, Foster City, CA) foram spray de 2,3 kV de tensão, temperatura da fonte de íons de 150 ° C, GS1 de 12, e gás de cortina 15. </li><li> A amostra é analisada por SRM / MRM, injetando 10 mL da amostra. Áreas dos picos são integrados para peptídeos leves e pesados ​​usando o Skyline programa. 14 Calcule a razão da área de pico (PAR) do peptídeo unlabeled / rotulados em cada amostra usando a transição mais abundante que é livre de ruído de fundo, neste caso, a transição y5 (peptídeo luz 476,3&gt; 579,3, pesado peptídeo padrão 481,3&gt; 589,3). </li></ol> 4. Resultados representativos: Medida razões das áreas de pico (em relação peptídeo luz endógena para peptídeo pesados ​​spiked isotopicamente marcado) fornecer uma medida quantitativa do peptídeo alvo. Figura 3 mostra um exemplo de cromatograma peptídeos leves e pesados ​​em uma amostra SISCAPA enriquecido. Note-se que os peptídeos leves e pesados ​​eluir no mesmo tempo e várias transições podem ser monitorados para cada peptídeo para confirmar a identidade. <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="Figura 1"/> Figura 1. Panorama Um esquema do processo SISCAPA. Uma mistura de proteínas complexo é digerido em peptídeos. Analitos peptídeo alvo (analito endógenos e uma spiked isótopo estável com etiqueta padrão interno) são enriquecidos com anticorpos anti-peptídeo imobilizada em proteína G-revestido partículas magnéticas. Após isolamento, os peptídeos são alvo eluída as partículas magnéticas e analisados ​​por espectrometria de massa para quantificação em relação ao padrão interno. <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="Figura 2"/> Figura 2. MS / MS espectro da GDSLAYGLR peptídeo mostrando seleção de três fragmentos de SRM / MRM transições. <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="Figura 3"/> Figura 3. Cromatogramas Exemplo mostrando o perfil de pico de um analito peptídeo de luz (vermelho) eo pesado isótopo estável com etiqueta padrão interno (azul). Cromatogramas para a transição y5 do peptídeo luz (476,3&gt; 579,3) e de isótopos estáveis ​​pesados ​​rotulados padrão (481,3&gt; 589,3) são plotados ao longo do tempo como eles saem da sistema de cromatografia.

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Não há conflitos de interesse declarados.

O passo mais crítico no protocolo, conforme descrito é garantir as contas permanecem bem misturado durante o período de incubação. Permitindo que as contas para se depositar no fundo do poço / frasco irá resultar em aumento da variabilidade. Também é importante para spin-down qualquer líquido que possa permanecer no topo do poço / frasco após o período de incubação. Digestão com tripsina reprodutíveis também é crítica. O procedimento para a digestão descrita aqui tem sido utilizada extensivamente em nosso laboratório em conjunto com SISCAPA, no entanto, é provável que os métodos alternativos de digestão pode ser otimizado para um determinado conjunto de proteínas-alvo 15 eluição do anticorpo livre não interfere com a detecção do. peptídeo, provavelmente porque o anticorpo é excluído da coluna ou elui mais tarde do que os peptídeos, mas o anticorpo pode ser reticulado às esferas G Protein antes de afinidade em experiências de enriquecimento de grande ou se anticorpo livre se torna um problema. Temos também que era útil para colocar um ímã sob a placa de amostra no amostrador automático, bem como lavar a coluna armadilha na direção contrária do fluxo (em comparação com a carga) para remover todas as pérolas residual ou partículas. Além da abordagem magnética talão descrito, a técnica também pode ser adaptado para um formato de coluna (ou seja, cromatografia de afinidade). 12, 16 Quando o usuário está confortável com o protocolo geral, a técnica também é passível de várias modificações que melhoram o ensaio geral. 11 Primeiro, é possível analisar analitos múltiplos em um único ensaio, combinando anticorpos na etapa de enriquecimento (multiplexação por exemplo). O espectrômetro de massa é capaz de analisar simultaneamente um grande número de analitos. Segundo, aumentando o volume de amostra original melhora a sensibilidade do ensaio.

<table border="1"><tbody><tr><th> Nome do material </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> 1000ul placas poço profundo de 96 poços </td><td> Eppendorf </td><td> 951032786 </td></tr><tr><td> Oasis HLB 1 cc placa do cartucho, Sep-pak, 40 mg placa de 96 poços </td><td> Águas </td><td> 186003966 </td></tr><tr><td> Kingfisher placa de 96 poços </td><td> Thermo Fisher Scientific </td><td> 97002540 </td></tr><tr><td> Claro de 96 poços, placa de parede branca </td><td> Bio-Rad </td><td> HSP9601 </td></tr><tr><td> Folha de cobertura para placa de 96 poços </td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> Axymat esteira de vedação para placa de 96 poços </td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> X furar filme </td><td> Sigma-Aldrich </td><td> Z722502 </td></tr><tr><td> Kingfisher processador magnético talão com PCR cabeça ímã </td><td> Thermo Fisher Scientific </td><td> HSP 9601 </td></tr></tbody></table> Tabela 1: Materiais <table border="1"><tbody><tr><th> Nome do material </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Uréia </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 (TDT) </td><td> Perfurar </td><td> 20291 </td><td> &quot;Não-pesar ditiotreitol&quot; </td></tr><tr><td> Iodoacetamida (IAM) </td><td> Sigma Aldrich </td><td> I1149 </td><td></td></tr><tr><td> Tripsina Ouro </td><td> Promega </td><td> V5280 </td><td></td></tr><tr><td> Proteína G esferas magnéticas, 2.8um </td><td> Invitrogen </td><td> 10004D </td><td></td></tr><tr><td> Tampão fosfato (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> Acetonitrila </td><td> Thermo Fisher Scientific </td><td> A998-1 </td><td></td></tr><tr><td> Ácido acético </td><td> Sigma Aldrich </td><td> 242853 </td><td></td></tr></tbody></table> Tabela 2: Reagentes <table border="1"><tbody><tr><th> Solução </th><th> Comentários (opcional) </th></tr><tr><td> 100 mM Tris pH 8 </td><td></td></tr><tr><td> 9 M de uréia / 30 mM DTT em 100 mM Tris (pH 8) </td><td> deve ser renovada a cada vez </td></tr><tr><td> IAM 500 mM em 100 mM Tris (pH 8) </td><td> deve ser renovada a cada vez </td></tr><tr><td> Tripsina 1mg/mL em 100 mM Tris pH 8 </td><td></td></tr><tr><td> Inibidor de tripsina 1mg/mL em 100 mM Tris pH 8 </td><td></td></tr><tr><td> Isótopo estável padrão Mastermix </td><td></td></tr><tr><td> Anti-peptídeo Mastermix anticorpos </td><td></td></tr></tbody></table> Tabela 3: Soluções para estar preparado

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

Há uma grande necessidade de ensaios quantitativos para medir proteínas. Imunoensaios sanduíche tradicional, amplamente considerado o padrão ouro na quantificação, estão associados a um alto custo, longo tempo, e estão repletas de desvantagens (por exemplo, anticorpos heterofílicos, a interferência auto-anticorpos, &#39;gancho-efeito &quot;). Uma técnica Uma alternativa é o enriquecimento de afinidade de peptídeos acoplado com espectrometria de massa quantitativos, comumente referido como SISCAPA (Padrões de Isótopos Estáveis ​​e Captura de Anticorpos Anti-Peptide). 2 Nesta técnica, a afinidade de enriquecimento de peptídeos com a diluição de isótopos estáveis ​​e detecção de selecionados / múltiplos de reação monitoramento espectrometria de massa ( SRM / MRM-MS) fornece medição quantitativa de peptídeos como substitutos para suas respectivas proteínas. SRM / MRM-MS está bem estabelecido para a quantificação precisa de pequenas moléculas 3, 4 e mais recentemente foi adaptado para medir as concentrações de proteínas no plasma e lisados ​​celulares. 5-7 Para alcançar quantificação de proteínas, estas moléculas maiores são digeridos para peptídeos componente usando uma enzima, como tripsina. Um ou mais peptídeos selecionados cuja seqüência é exclusivo para a proteína-alvo, em que as espécies (ou seja, &quot;proteotypic&quot; peptídeos) são, então, enriquecido a partir da amostra usando anticorpos anti-peptídeo e medido como substitutos estequiométrica quantitativa para a concentração de proteína na amostra. Assim, acoplado a diluição de isótopos estáveis ​​(SID) (ou seja, um spiked-in isótopo estável rotulados peptídeo standard), SRM / MRM podem ser usados ​​para medir as concentrações de peptídeos proteotypic como substitutos para a quantificação de proteínas em matrizes biológicas complexas. Métodos Os ensaios têm várias vantagens em comparação com imunoensaios tradicional. Os reagentes são relativamente menos caro para gerar, a especificidade para o analito é excelente, os ensaios podem ser altamente multiplexados, de enriquecimento pode ser realizado a partir do plasma puro (sem esgotamento necessário), ea técnica é passível de uma grande variedade de proteínas ou modificações de interesse. 8-13 Neste vídeo podemos demonstrar o protocolo básico, adaptado a uma plataforma magnética talão.

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

Padrões de Isótopos estáveis ​​e Captura de Anticorpos Anti-Peptide (SISCAPA) casais enriquecimento afinidade de peptídeos com isótopos estáveis ​​de diluição espectrometria de massa (MRM-MS) para fornecer a medição quantitativa de peptídeos como substitutos para suas respectivas proteínas. Aqui nós descrevemos o protocolo usando partículas magnéticas em um formato parcialmente automatizados.

Quantificação de Proteínas Usando Enriquecimento imunoafinidade Peptide Acoplado com Espectrometria de Massa

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 visualizações.  Fred Hutchinson Cancer Research Center - FHCRC. Padrões de Isótopos estáveis ​​e Captura de Anticorpos Anti-Peptide (SISCAPA) casais enriquecimento afinidade de peptídeos com isótopos estáveis ​​de diluição espectrometria de massa (MRM-MS) para fornecer a medição quantitativa de peptídeos como substitutos para suas respectivas proteínas. Aqui nós descrevemos o protocolo usando partículas magnéticas em um formato parcialmente automatizados.

Vídeo: 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 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

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.

A avaliação quantitativa do Lipidome fermento usando Electrospray Espectrometria de Massa de Ionização

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.

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 ...

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.

Em Quantificação in vivo de proteínas G Interações Receptor Acoplado usando espectral Resolvido Microscopia de dois fótons

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.

Perfil de metiltransferases e Outros S-Adenosil- L Homocisteína-Proteínas de Ligação de captura por Espectrometria de Massa Compound (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 ...

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 ...

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.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

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 de Espectrometria de Massa Compound - uma ferramenta poderosa para identificar novos c-di-GMP efetoras 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.

Reação selecionada Monitoramento Espectrometria de Massa para Absolute Quantificação de Proteína

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.

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 ...