Este trabalho foi financiado pelo Human Frontier Organization Programa Ciência (HFSP Award 2007, RGP0058/2007-C). Agradecemos ao Prof Richard Roberts para o início do projeto e para discussões frutíferas.

<ol>
	<li>Katayama, H., Oda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+proteomics+for+drug+discovery+based+on+compound-immobilized+affinity+chromatography.">Chemical proteomics for drug discovery based on compound-immobilized affinity chromatography.</a> J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 855, 21-27 (2007).</li><li>Barglow, K. T., Cravatt, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+protein+profiling+for+the+functional+annotation+of+enzymes.">Activity-based protein profiling for the functional annotation of enzymes.</a> Nat. Methods. 4, 822-827 (2007).</li><li>Koster, H., Little, D. P., Luan, P., Muller, R., Siddiqi, S. M., Marappan, S., Yip, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capture+compound+mass+spectrometry:+a+technology+for+the+investigation+of+small+molecule+protein+interactions.">Capture compound mass spectrometry: a technology for the investigation of small molecule protein interactions.</a> Assay Drug Dev. Technol. 5, 381-390 (2007).</li><li>Dalhoff, C., Hueben, M., Lenz, T., Poot, P., Nordhoff, E., Koster, H., Weinhold, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+S-Adenosyl-L-homocysteine+Capture+Compounds+for+Selective+Photoinduced+Isolation+of+Methyltransferases.">Synthesis of S-Adenosyl-L-homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases.</a> ChemBioChem. 11, 256-265 (2010).</li><li>Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine.">S-Adenosylmethionine.</a> Int. J. Biochem. Cell. Biol. 32, 391-3952 (2000).</li><li>Cantoni, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+methylation:+selected+aspects.">Biological methylation: selected aspects.</a> Annu. Rev. Biochem. 44, 435-451 (1975).</li><li>Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Watson+and+Crick:+DNA+methylation+and+molecular+enzymology+of+DNA+methyltransferases.">Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases.</a> ChemBioChem. 3, 274-293 (2002).</li><li>Chow, C. S., Lamichhane, T. N., Mahto, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+nucleotide+repertoire+of+the+ribosome+with+post-transcriptional+modifications.">Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications.</a> ACS Chem. Biol. 2, 610-619 (2007).</li><li>Grillo, M. A., Colombatto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine+and+protein+methylation.">S-Adenosylmethionine and protein methylation.</a> Amino Acids. 28, 357-362 (2005).</li><li>Fujioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+small+molecule+methyltransferases:+their+structural+and+functional+features.">Mammalian small molecule methyltransferases: their structural and functional features.</a> Int. J. Biochem. 24, 1917-1924 (1992).</li><li>Borchardt, R. T., Salvatore, F., Borek, E., Zappia, V., Williams-Ashman, H. G., Schlenk, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biochemistry of S-Adenosylmethionine. , 151-171 (1977).</li><li>Hoffman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+analysis+of+the+chiral+and+covalent+instability+of+S-adenosyl-l-methionine.">Chromatographic analysis of the chiral and covalent instability of S-adenosyl-l-methionine.</a> Biochemistry. 25, 4444-4449 (1986).</li><li>Keller, A., Nesvizhskii, A. I., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirical+statistical+model+to+estimate+the+accuracy+of+peptide+identifications+made+by+MS/MS+and+database+search.">Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.</a> Anal. Chem. 74, 5383-5392 (2002).</li><li>Nesvizhskii, A. I., Keller, A., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+statistical+model+for+identifying+proteins+by+tandem+mass+spectrometry.">A statistical model for identifying proteins by tandem mass spectrometry.</a> Anal. Chem. 75, 4646-4658 (2003).</li></ol>

ThomasLenz, OliviaGrabner, MirkoGlinski, MathiasDreger e HubertKosterare empregados por caprotec Bioanalítica GmbH, Inc, que produz alguns dos reagentes utilizados neste artigo.

As seguintes precauções e comentários podem ser úteis quando, seguindo o protocolo descrito: a) A vantagem principal de CCs reside na formação de uma ligação covalente entre o CC eo MTase, pois isso permite subseqüentes condições de lavagem rigorosa. A reticulação covalente é realizado por um fotorreação desencadeada pela luz UV (310 nm max.). Luz do teto normal contém apenas uma pequena fração dos raios UV, no entanto, proteger a HAS CC-de maior tempo de exposição à sobrecarga ou mesmo a luz sol a irradiação controlada e de refrigeração na caproBox. b) As amostras biológicas de que o MTases devem ser isolados podem conter proteínas propensas a desnaturação, conseqüentemente, é obrigatório manter as amostras legal e para evitar a formação de espuma em todos os momentos. c) O caproBox esfria as amostras para 0-4 ° C, as lâmpadas que emitem a luz UV, no entanto, também emitem calor. Portanto, é necessário fazer uma breve centrifugação os frascos antes da irradiação, de modo proteínas aderindo à tampas de frasco ou paredes não pode formar sementes precipitação. d) Se re-suspensão das esferas magnéticas (por exemplo, as soluções de lavagem) não é possível com a mão, logo aplicar ultra-som, colocando as amostras em banho de ultra-som. e) Freshly preparar a solução ACN 0,2% TFA/60%. Descobrimos que de outra forma as proteínas capturados não pode ser clivada dos grânulos. f) A análise final das proteínas capturado é realizado por LC-MS/MS. Espectrometria de massa é um método altamente sensível. É necessário o uso de reagentes exclusivamente LC-MS grau nas etapas finais (passo 2.11 e mais). Evitar a contaminação dos experimentos por fontes de proteína externa, por exemplo, a queratina provenientes de poeira ou do experimentador. Particularmente durante as etapas finais da digestão, recomenda-se a prestar atenção a um espaço de trabalho limpo, usar luvas e um casaco de laboratório e, possivelmente, uma rede de cabelo ou, idealmente, realizar as etapas finais em uma bancada limpa. Prepare a 50 mM tampão bicarbonato de amônio usado para digerir tríptica em água LC MS grau, filtrar através de 0,22 mM, armazenar alíquota de filtro, a -20 ° C, e usar apenas uma vez cada alíquota para evitar contaminações. A solução de tripsina (seqüenciamento grau, Roche, preparar um 0,5 mg / mL solução pela adição de 1 mM HCl para liofilizado tripsina) pode ser armazenado a 4 ° C por várias semanas. g) Para obter espectros de massa de confiança é essencial para ter um spray estável em análise ESI-MS/MS. h) Para outros sistemas LC-MS/MS do que o utilizado no presente estudo, parâmetros de medição e algoritmos de identificação de peptídeos devem ser ajustadas individualmente. As seguintes modificações são possíveis com relação ao protocolo descrito: a) Como alternativa para liberar as proteínas dos grânulos em 60% TFA ACN/0.2% (passo 2.11), as proteínas podem ser diretamente tryptically digerida dentro de uma suspensão de esferas (volumes mesma no passo 5.1) ou, por SDS-PAGE, as proteínas podem ser liberados, suspendendo as contas de aquecimento e coletadas após a etapa 2,9-95 ° C por 10 min em tampão de amostra SDS (ambos, toda a suspensão ou apenas o sobrenadante pode ser carregado no bolso gel). Descobrimos que os grânulos liberam lentamente pequenas quantidades de polímeros em solução aquosa digerir tríptica durante on-bead digerir tríptica mesmo após várias etapas de lavagem aquosa. A contaminação interfere com polímero MS identificação de peptídeos e pode ser lavado as contas com 80% ACN (pelo menos três vezes). Após as etapas de lavagem de 80% ACN, as contas devem ser lavadas uma vez com água antes de on-bead digerir tríptica. b) Western blots utilizando estreptavidina-peroxidase e substrato horseraddish ECL também pode ser usado para visualizar crosslinking bem-sucedida do CC contendo biotina às proteínas. Portanto, ou o gel obtido após a etapa 3.2 pode ser apagado ou, porque a sensibilidade é cerca de 10 vezes maior do que a coloração de prata de géis, 10 amostras mL após a etapa 2,7 também podem ser analisados ​​por western blot. Lembre-se que neste último caso proteínas endogeneously biotinilado também será detectada além de proteínas artificialmente biotinilado pela HAS-CC. c) Descobrimos que depende do sistema especial (lisado, dirigida proteínas-alvo, seletividade e reatividade da função que ele CC), se o &quot;off-talão&quot; configuração, onde a reação de reticulação ocorre entre CC livre e proteínas em solução 4 ou o momento descrito &quot;on-talão&quot; configuração (Figura 1B) tem melhor desempenho. Em geral, o método também deve ser compatível com qualquer estado-da-arte da proteína isótopo estável ou tecnologia rotulagem peptídeo, ou avaliação da amostra captura por eletroforese em gel 2D. No &quot;off-talão&quot; de configuração, também é possível capturar proteínas dentro das células inteiras (dados não publicados). Além disso, a droga ou site cofactor ligação de uma proteína pode ser delineado por determinar a posição de reticulação bem perto do CC dentro da seqüência de proteínas por MS seqüenciamento de peptídeos. O modo de ligação de uma pequena molécula de uma proteína pode ser explorado usando diferentes chemical posições de anexação na função de seletividade e comprimentos diferentes linker. Como mostrado no presente estudo, os parceiros também proteína de ligação das proteínas dirigida pela função de seletividade (RplK como substrato de PRMA) ou desconhecidas interações pequena molécula de proteína (HAS para GINA) podem ser identificados. Resumida, a característica adicional do CCs, a reatividade foto crosslinking, permite o isolamento e identificação de proteínas de baixo abundante ou famílias de proteínas funcionais a partir de misturas complexas de proteínas com alta sensibilidade e fornece aos cientistas uma ferramenta adicional para o estudo de moléculas pequenas - interações proteína .

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>SAH caproKit&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-1010-050 (50 reactions) 1-1010-010 (10 reactions)</td>
<td>Includes the SAH-CC, SAH competitor, streptavidin coated magnetic beads, capture buffer, and wash buffer</td>
</tr>
<tr>
<td>caproBox&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5010-003 (110 V) 1-5010-004 (230 V)</td>
<td>For reproducible photo-activation while cooling the samples</td>
</tr>
<tr>
<td>caproMag&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5100-001</td>
<td>For easy handling of magnetic particles without pipetting</td>
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profiling of methyltransferases and other s-adenosyl-l-homocysteine-binding proteins by capture compound mass spectrometry (ccms)

Há uma variedade de abordagens para reduzir a complexidade do proteoma com base na pequena molécula de proteína funcional de interações tais como cromatografia de afinidade ou de um perfil a atividade da proteína Baseado 2. Compostos de Captura trifuncional (CCs, Figura 1A) 3 são a base para uma abordagem genérica, em que o equilíbrio inicial-driven interação entre uma sonda pequena molécula (a função de seletividade, aqui S-adenosil-L-homocisteína, HAS, Figura 1A) e proteínas-alvo é irreversível fixos foto crosslinking entre uma função de reatividade independente foto-que podem ser activados (aqui um phenylazide) do CC e da superfície das proteínas-alvo. A função de triagem (aqui biotina) serve para isolar o CC - conjugados de proteínas do complexo misturas biológica com a ajuda de uma fase sólida (grânulos aqui estreptavidina magnética). Duas configurações dos experimentos são possíveis: &quot;off-talão&quot; 4 ou o momento descrito &quot;on-talão&quot; configuração (Figura 1B). A função de seletividade pode ser praticamente qualquer molécula pequena de interesse (substratos, inibidores, moléculas da droga). S-adenosil-L-metionina (SAM, Figura 1A) é, provavelmente, segundo a ATP, o co-fator mais utilizado na natureza 5, 6. É usado como o principal doador de metila grupo em todos os organismos vivos com a reação química pode ser catalisada pela SAM-dependente metiltransferases (MTases), que metilato de DNA 7, 8 RNA, proteínas 9, ou pequenas moléculas 10. Dado o papel crucial das reações de metilação em diversos cenários fisiológicas (regulação de genes, a epigenética, o metabolismo), o perfil dos MTases se pode esperar para se tornar de importância similar em proteômica funcional como o perfil de quinases. Ferramentas analíticas para o seu perfil, no entanto, não estavam disponíveis. Recentemente, introduziu um CC com HAS como grupo seletividade para preencher esta lacuna tecnológica (Figura 1A). HAS, o produto da SAM, após a transferência de metila, é um inibidor produto conhecido geral MTase 11. Por esta razão e porque o natural cofactor SAM é usado pelas enzimas ainda a transferência de outras partes do cofator ou iniciar reações radicais, bem como devido à sua instabilidade química, 12 de HAS é uma função ideal para uma seletividade como alvo MTases CC. Aqui, relatamos a utilidade da HAS-CC e CCMS por MTases profiling e outros HAS proteínas de ligação a partir do DH5α estirpe de Escherichia coli (E. coli), um dos melhores caracterizados os procariontes, que tem servido como o modelo preferido organismo em inúmeros estudos bioquímicos, biológicos e biotecnológicos. Foto-ativada reticulação aumenta a produtividade e sensibilidade do experimento, a especificidade e pode ser facilmente testada em experimentos de competição para usar um excesso de HAS livre.

Watch this Scientific Journal Video about Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS at JoVE.com

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

Compostos de captura são trifuncional pequenas moléculas para reduzir a complexidade do proteoma por funcionais reversíveis interação molécula de proteína pequena seguido por foto-reticulação e purificação. Aqui usamos um Composto de captura com<em&gt; S</em&gt;-Adenosil-<sub&gt; L</sub&gt;-Homocisteína vinculativo, como função de seletividade para isolar metiltransferases a partir de um<em&gt; Escherichia coli</em&gt; Lisado de células inteiras e identificá-los pelo 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-methyltransferases-other-s-adenosyl-l-homocysteine-binding

Research

JoVE Journal

Biology

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

15.5K visualizações.  caprotec bioanalytics GmbH. Compostos de captura são trifuncional pequenas moléculas para reduzir a complexidade do proteoma por funcionais reversíveis interação molécula de proteína pequena seguido por foto-reticulação e purificação. Aqui usamos um Composto de captura com S-Adenosil- L-Homocisteína vinculativo, como função de seletividade para isolar metiltransferases a partir de um Escherichia coli Lisado de células inteiras e identific...

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

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Perfil de anticorpos por sistemas de imunoprecipitação luciferase (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

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 ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Oligo Profiling Massa (OLIMP) de polissacarídeos extracelulares

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

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 ...

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 ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

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 ...

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 ...

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 ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Rotulagem específica de seqüência de ácidos nucléicos e proteínas com Metiltransferases e cofator Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...