Este trabalho é apoiado em parte do National Institutes of Health Grant GM-26738 (para JAT).

1. PAK2 Ativação <ol><li> Adicione a quantidade pré-testados apropriado de caspase 3-20 mL de 12 mg / ml em tampão PAK2 A e (50 mM DTT mM Tris-HCl, 150 mM NaCl, e 2 em pH 7,5) incubar a 34 ° C por 30 min plenamente decompor e PAK2 ativar. </li><li> Analisar os produtos de dissociação por SDS-PAGE (10%) e coloração Coommassie azul. </li><li> Adicione o PAK2 clivada para a ativação de buffer B (50 mM Tris-HCl, 150 mM NaCl, 15 mM MgCl 2, 10 mM ATP) em pH 7,5, em 20 l para uma concentração final de 10 mg / ml PAK2, e incubar a 34 ° C por 30 min para ativar completamente PAK2 por autofosforilação em 8 sites. </li><li> Confirmar clivagem cheio de PAK2 pela migração do p34 e p27 fragmentos como determinado por SDS-PAGE 10. Verificação adequada das condições autofosforilação é feita através de autoradiografia em um experimento separado usando (32 P) ATP ou espectrometria de massa para detectar fosfopeptídeos. </li></ol> 2. Identificaçãode pepsina-digerida PAK2 Fragments <ol><li> Adicionar 1 ml de 0,1% de ácido Trifluroacetic (TFA) em pH 2,5 para lavar as contas agarose duas vezes antes de usar. Então, centrifugue as amostras por 15 segundos a 2.000 xg para remover as contas. </li><li> Adicionar PAK2 (20 mg em 2 mL de 150 mM NaCl, 50 mM DTT mM Tris-HCl e 2, pH 7,5) para 100 l de TFA 0,1%, e incubar a amostra por 5 min com 30 mL de contas pepsina-conjugado de agarose . </li><li> Equilibrar uma coluna de HPLC de fase reversa C18 com um sistema primário de solvente de TFA 0,1% em pH 2.5. O resumo da pepsina (50 ul) é eluída usando um gradiente de 100 min-linear de 0-80% de acetonitrila contendo TFA 0,1% a uma vazão de 0,2 ml / min. Frações são coletadas a cada 2 min. </li><li> Seca as frações de HPLC com uma velocidade-vac (3 h) e ressuspender as amostras em uma solução de acetonitrila (5 mL) e TFA 0,1% (5 mL), pH 2.5. </li><li> Inicialmente, cada fração é testado para peptídios usando o MALDI-TOF PerSeptive Biosystems Voyager STR DE (PE Biosystems, Foster City, CA, EUA). </li><li> As 16 frações contendo peptídeos são submetidos a espectrometria de massa em tandem utilizando um espectrômetro de massa Q-TOF e um QStar XL oMALDI MS / MS para identificar os fragmentos. </li><li> A seqüência de cada peptídeo é verificado pelos íons produto gerado pela espectrometria de massa em tandem com a teórica m / z relações com base na seqüência primária de PAK2 usando a Protein Prospector website ( <a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ) 11. </li></ol> 3. Medição de amida H / D Câmbio <ol><li> Mantenha todas as soluções e reagentes necessários para H / D de câmbio em 25 ° C em banho-maria. ATP é dissolvido em água eo pH é ajustado para 7,0 antes da utilização. O adicional de 4 mM ATP na 2 D O tampão é para prevenir ATP dissociada PAK2 após a diluição. </li><li> Iniciar H / D de troca através da adição de 18 mL de tampão D 2 O (50 mM pH 6,98 MOPS, 125 mM NaCl) a 2 mL (20 mg) De PAK2 em um tampão contendo 150 mM NaCl, 50 mM DTT mM Tris-HCl (pH 7,5) e 2, resultando em um nível PAK2 de 10 mg / ml e um pH de 7,0 a 0 ° C. </li><li> Incubar as amostras em triplicata na troca H / D de 0, 0,5, 1, 3, 5 e 10 min, ou 24 h. </li><li> Extinguir o H / D processo de troca, adicionando 180 mL de gelada TFA 0,1% em pH 2.2, que traz o pH para 2,5 em um volume final de 200 mL. Para as amostras contendo ATP, 180 mL de gelo frio TFA 0,11% em pH 2.2 é utilizado para manter o pH de 2,5 na condição saciada. </li><li> Adicionar uma alíquota (100 l) de cada amostra a 30 mL saciada de contas ativadas pepsina-conjugado de agarose. A digestão pepsina é permitido proceder em gelo por cinco minutos e as amostras são centrifugadas a cada 30 seg. </li><li> Terminar a digestão por centrifugação da amostra por 15 segundos a 5.000 xg a 4 ° C para remover a pepsina. </li><li> Rapidamente congelar as amostras em N 2 líquido e armazenar a -80 ° C para não mais de 2dias antes da MALDI-TOF análise. </li><li> A matriz para MALDI é de 5 mg / ml de α-ciano-4-hidroxicinâmicos ácido (CHCA) dissolvido em uma solução (1:1:1) de etanol, acetonitrila e TFA 0,1% em pH 2,5 e mantidos a 0 ° C. </li><li> Parcialmente descongelar cada amostra rapidamente e misture 1 ml de cada amostra com 1 ml de matriz e local em um 4 ° C placa-alvo MALDI. </li><li> Aplicar um vácuo moderado ao prato para secar os pontos em 30 segundos antes de MALDI-TOF análise. O tempo entre o descongelamento da amostra e recuperação de espectro é de cerca de 3 min. </li><li> Adquirir MALDI-TOF espectros de massa com um Voyager Biosystems PerSeptive STR DE. Aquisição de dados em uma taxa de amostragem de 2 GHz a 100 mil canais de dados, com uma tensão de 20.000 V de aceleração, e uma tensão de rede usando 65% de extração atrasada com um atraso de pulso de 180 ns. Cem exames são em média de 2 min. O instrumento MALDI-TOF não se restringe a qualquer marca ou qualquer outro software específico. </li><li> Usar o Data Explorer programa de computador 4.0 para calculcomeu o deuteração de cada fragmento. </li><li> Calcular o câmbio de volta com base na razão entre o número deuteron real incorporado em 24 hr de H / D troca dividido por todos os possíveis locais de troca, que são a espinha dorsal amidas exceto a amida N-terminal (Figura 1). </li></ol> 4. Resultados representativos: A clivagem caspase e autofosforilação são verificados por SDS-PAGE coloração Commassie. PAK2 inativos é uma banda única de 58 kDa. Clivagem da caspase PAK2 está completa, e produz dois fragmentos, p27 e p34 que migram separadamente. A migração de p27 e p34 autofosforilada mostra uma sobreposição no gel devido à migração retardada do p27 totalmente fosforilados fragmento (Figura 2). Experimentos H / D de câmbio são realizadas várias vezes entre 0 e 10 min (Figura 3). Como exemplo, o tempo de incorporação de deutério na m / zpeak 1.697,85 de PAK2 inativo é composto de vários picos de isótopos, como mostrado pela mudança do envelope de massa ao longo do tempo. <img alt="Figura 1" src="/files/ftp_upload/3602/3602fig1.jpg" /> Figura 1. Equação para o cálculo da deuteração. <img alt="Figura 2" src="/files/ftp_upload/3602/3602fig2.jpg" /> Figura 2. Autofosforilação e clivagem caspase de PAK2. Caspase clivada PAK2 (faixa esquerda), PAK2 inativos intacta (meio lane) e caspase clivada PAK2 autofosforilada (pista direita) foram analisadas por SDS-PAGE e coloração Coommassie azul. Adaptado de Hsu, YH et al 2. <img alt="Figura 3" src="/files/ftp_upload/3602/3602fig3.jpg" /> Figura 3. Espectro de massa de um curso do tempo de incorporação de deutério para PAK2 inativo. PAK2 inativos foi submetido a H / D exchaESL para 00-10 min e analisados ​​por MALDI-TOF. Um exemplo da distribuição expandida isotópica de MALDI-TOF de pico m / z 1.697,85 durante o curso do tempo de H / D de câmbio é mostrado. A linha vermelha pontilhada indica a média do envelope de massa e da mudança do envelope de massa mostra o deuteração de um peptídeo.

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 283, 9820-9827 (2008).</li><li>Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hPAK65+by+caspase+cleavage+induces+some+of+the+morphological+and+biochemical+changes+of+apoptosis.">Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13642-13647 (1997).</li><li>Rudel, T., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+morphological+changes+in+apoptotic+cells+regulated+by+caspase-mediated+activation+of+PAK2.">Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.</a> Science. 276, 1571-1574 (1997).</li><li>Roig, J., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytostatic+p21+G+protein-activated+protein+kinase+gamma-PAK.">Cytostatic p21 G protein-activated protein kinase gamma-PAK.</a> Vitam. Horm. 62, 167-198 (2001).</li><li>Walter, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+and+activation+of+p21-activated+protein+kinase+gamma-PAK+by+CPP32+(caspase+3).+Effects+of+autophosphorylation+on+activity.">Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.</a> J. Biol. Chem. 273, 28733-28739 (1998).</li><li>Clauser, K. R., Baker, P., Burlingame, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+accurate+mass+measurement+(+/-+10+ppm)+in+protein+identification+strategies+employing+MS+or+MS/MS+and+database+searching.">Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.</a> Anal. Chem. 71, 2871-2882 (1999).</li></ol>

Não há conflitos de interesse declarados.

Identificação dos fragmentos Pepsin-digerida é uma etapa crítica da experiência de troca H / D. Identificação incorreta do peptídeo pode levar a uma conclusão errada. PAK2 pepsina-digerida é eluído através de uma coluna de fase reversa HPLC C18 para obter um fundo limpo. Em nossos experimentos, um total de 40 frações foram coletadas e submetidas a MALDI-TOF. Peptídeos múltiplos puderam ser identificados em cada uma das frações. Os peptídeos foram submetidos a espectrometria de massa (Q-TOF MS / MS e oMALDI MS / MS) para identificar suas seqüências. Os espectros MS / MS de um peptídeo deve ter pelo menos três íons produto para confirmar a identificação do peptídeo. O Data Explorer programa de computador 4.0 (Applied Biosystems) realiza a correção inicial, e filtragem de ruído. Cada espectro deve ser calibrado com base em dois picos seqüenciado. Nós calibrar nosso espectro pela massa teórica do undeuterated m / z que são 923,45 e 1.697,84. TEle precisão massa após a calibração pode chegar a 10 ppm. Após deuteração, o mono-isótopos podem se deslocar para uma maior massa. Aumenta o passo unitário para estes índices m / z rendeu as massas melhorada associada deuteração. As intensidades de pico do envelope de massa não afetará o nível deuteração. No entanto, o pico de intensidade dos picos de isótopos em um envelope de massa específica são fundamentais para o cálculo da massa média. O primeiro passo do cálculo do deutério é incorporada subtraindo a massa de peptídeos da amostra não-deuterado a partir da amostra deuterado. Três outros fatores, a diluição D 2 O, os dêuterons residual nas cadeias laterais eo intercâmbio de volta, terá de ser ainda considerados no cálculo (Figura 1). A D 2 O diluição é o fator de diluição de D 2 O após a mistura da amostra e D 2 O tampão para iniciar a troca H / D. O deutério residual (4,5%) nas cadeias laterais da pepsina-digestpeptídeos ed precisa ser subtraído. A parte de trás de troca é a perda inevitável de deuteração em todos os peptídeos deuterado e pepsina-digerida no processo de medição de massa. O peptídeo mais acessíveis solvente (m / z = 1.105,60) após 24 horas de H / D de câmbio representa uma região deuteração completo. O número deuteron incorporados para cada um dos peptídeos é a diferença entre o centróide do deuterado e não deuterado peptídeos péptica PAK2. O peptídeo mais altamente deuterado m / z 1105 foi selecionado para representar deuteração cheia quando PAK2 foi de 24 h de H / D de câmbio. A Relação de Troca Voltar foi calculado pelo número deuteron real incorporado em 24 hr de H / D troca dividido por todos os possíveis locais de troca de peptídeo m / z 1105.

<table border="1"></table><table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> D 2 O </td><td> Aldrich </td><td> 7789-20-0 </td><td></td></tr><tr><td> α-ciano-4-ácido hidroxicinâmicos </td><td> Sigma </td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI-TOF </td><td> PE Biosystems </td><td> Voyager STR DE </td><td></td></tr><tr><td> Q-TOF espectrômetro de massa </td><td> Q-TOF Ultima-Global </td><td></td><td></td></tr><tr><td> QStar XL oMALDI MS / MS </td><td> Applied Biosystems </td><td></td><td></td></tr><tr><td> Data Explorer programa de computador 4,0 </td><td> Applied Biosystems </td><td></td><td></td></tr&gt; </tbody></table>

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

Troca hidrogênio / deutério amida (H / D de câmbio) acoplado com espectrometria de massa tem sido amplamente utilizado para analisar a interface de interações proteína-proteína, mudanças conformacionais de proteínas, dinâmica de proteína e proteína-ligante interações. H / D de câmbio sobre as posições amida espinha dorsal tem sido utilizada para medir as taxas de deuteração do micro-regiões em uma proteína por espectrometria de massa 1,2,3. A resolução deste método depende da digestão da proteína pepsina deuterado de interesse em peptídeos que, normalmente, faixa 3-20 resíduos. Embora a resolução de H / D de troca medido por espectrometria de massa é menor do que a resolução resíduo única medida pelo único método Quantum Coherence heteronucleares (HSQC) da RMN, a medição de espectrometria de massa em H / D de câmbio não é limitada pelo tamanho da proteínas 4. H / D de câmbio é realizada em uma solução aquosa que mantém a conformação da proteína. Nós fornecemos um método que utilizes o MALDI-TOF para a detecção de dois, em vez de um HPLC / ESI (ionização electrospray)-MS sistema 5,6. A MALDI-TOF fornece dados precisos intensidade de massa para os peptídeos da proteína digerida, neste caso, a proteína quinase PAK2 (também chamado de γ-Pak). Proteólise de Pak 2 é realizado em uma digestão pela pepsina offline. Este método alternativo, quando o usuário não tem acesso a uma coluna de HPLC e pepsina conectado a espectrometria de massa, ou quando a coluna de pepsina em HPLC não resultar em um mapa melhor digestão, por exemplo, o bissulfeto-ligados fortemente secretado Fosfolipase A 2 (SPLA 2). Utilizando este método, conseguimos monitoradas as mudanças no nível deuteração durante a ativação da caspase PAK2 por clivagem 3 e autofosforilação 7,8,9.

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

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

MALDI-TOF espectrometria de massa foi utilizado com sucesso para monitorar a troca hidrogênio / deutério amida em proteína quinase PAK2 ativação.

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 ...

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17.7K visualizações.  Tunghai University. MALDI-TOF espectrometria de massa foi utilizado com sucesso para monitorar a troca hidrogênio / deutério amida em proteína quinase PAK2 ativação.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

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

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

MALDI 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 ...

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

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

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

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

Uso de MALDI-TOF Espectrometria de Massa e um banco de dados personalizado para caracterizar bactérias indígenas para uma caverna ambiente único (Kartchner Caverns, AZ, EUA)

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

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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

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

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 ...

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 ...

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

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

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

Fluxo de trabalho completo para Análise de histona modificações pós-traducionais Usando Bottom-up Espectrometria de Massa: De histona Extração de Análise de Dados

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