Ce travail a été soutenu par l&#39;Organisation Human Frontier Science Program (HFSP Award 2007, RGP0058/2007-C). Nous tenons à remercier le professeur Richard Roberts pour amorcer le projet et pour des discussions fructueuses.

<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 et HubertKosterare employées par caprotec bioanalytique GmbH, Inc qui produit certains des réactifs utilisés dans cet article.

Les précautions suivantes et les commentaires peuvent être utiles quand on suit le protocole décrit: a) Un avantage majeur du CC réside dans la formation d&#39;une liaison covalente entre le CC et le MTase, car cela permet subséquente des conditions de lavage rigoureux. La réticulation covalente est réalisée par un photoréaction déclenchée par la lumière UV (310 nm max.). Plafonnier normal contient seulement une petite fraction des rayons UV, toutefois, de protéger le SEP-CC de plus l&#39;exposition au rétroprojecteur ou la lumière, même lever du soleil, à l&#39;irradiation contrôlée et refroidi dans le caproBox. b) Les échantillons biologiques à partir desquels l&#39;MTases doivent être isolés peuvent contenir des protéines susceptibles de dénaturation, par conséquent, il est obligatoire de conserver les échantillons frais et d&#39;éviter de faire mousser à tout moment. c) Le caproBox refroidit les échantillons à 0-4 ° C, les lampes émettant de la lumière UV, cependant, émettent également de la chaleur. Par conséquent, il est nécessaire de centrifuger brièvement les flacons avant l&#39;irradiation, donc des protéines en respectant les plafonds ou les murs flacon ne peuvent pas former des graines de précipitation. d) Si la remise en suspension des billes magnétiques (par exemple dans les solutions de lavage) n&#39;est pas possible à la main, peu applicables à ultrasons en plaçant les échantillons dans un bain à ultrasons. e) fraîchement préparer la solution d&#39;ACN 0,2% TFA/60%. Nous avons constaté que les protéines capturées contraire ne peut être clivée de la perles. f) L&#39;analyse finale des protéines capturées est réalisée par LC-MS/MS. La spectrométrie de masse est une méthode hautement sensible. Il est nécessaire d&#39;utiliser des réactifs exclusivement LC-MS de qualité dans les étapes finales (étape 2.11 et plus). Eviter la contamination des expériences par des sources de protéines externes, la kératine, par exemple en provenance de la poussière ou de l&#39;expérimentateur. Particulièrement au cours des étapes de digestion finale, il est recommandé de faire attention à un espace de travail propre, de porter des gants et une blouse de laboratoire et, éventuellement, un filet à cheveux ou idéalement exécuter les étapes finales sous un banc propre. Préparer le tampon de 50 mM de bicarbonate d&#39;ammonium utilisé pour digestion trypsique en LC MS eau de qualité, filtrer à travers filtre de 0,22 um, aliquote, conserver à -20 ° C, et utiliser chaque aliquote qu&#39;une seule fois pour éviter les contaminations. La solution de trypsine (séquençage de grade, Roche, préparer un g 0,5 / ul solution en ajoutant HCl 1 mM d&#39;lyophilisé trypsine) peuvent être conservés à 4 ° C pendant plusieurs semaines. g) Pour obtenir des spectres de masse fiable, il est essentiel d&#39;avoir un spray stable dans l&#39;analyse ESI-MS/MS. h) Pour les systèmes LC-MS/MS autre que celui utilisé dans la présente étude, les paramètres de mesure et d&#39;algorithmes d&#39;identification de peptides doivent être ajustés individuellement. Les modifications suivantes sont possibles en ce qui concerne le protocole décrit: a) Comme alternative à la libération des protéines à partir des billes par du TFA 60% ACN/0.2% (étape 2.11), les protéines peuvent être directement tryptically digérés dans un suspension de billes (mêmes volumes que à l&#39;étape 5.1) ou, pour SDS-PAGE, les protéines peuvent être libérés par la suspension et le chauffage des perles recueillies après l&#39;étape de 2,9 à 95 ° C pendant 10 min dans du tampon échantillon de SDS (à la fois, la suspension totale ou seulement le surnageant peut être chargé dans la poche de gel). Nous avons constaté que les billes de libérer lentement de petites quantités de polymère dans la solution aqueuse digérer tryptique cours sur billes digérer tryptique même après plusieurs étapes de lavage aqueux. La contamination de polymères interfère avec MS peptide d&#39;identification et peuvent être lavés les perles avec 80% d&#39;ACN (au moins trois fois). Après les étapes de lavage de 80% d&#39;ACN, les billes doivent être lavés une fois avec de l&#39;eau avant de le digérer tryptique-billes. b) Western blot à l&#39;aide de streptavidine-peroxydase et le substrat horseraddish ECL peut également être utilisé pour visualiser réticulation succès du CC biotine contenant les protéines. Par conséquent, soit le gel obtenu après l&#39;étape 3.2 peut être effacé ou, parce que la sensibilité est environ 10 fois plus élevé que coloration à l&#39;argent de gels, de 10 échantillons ul après l&#39;étape 2.7 peut également être analysée par western blot. L&#39;esprit que dans ce dernier cas protéines biotinylées endogeneously seront également détectés en dehors des protéines biotinylées artificiellement par le SEP-CC. c) Nous avons constaté que cela dépend du système spécial (lysat, adressée protéines cibles, la sélectivité et la fonction qu&#39;il réactivité de CC), si le &quot;hors-talon&quot; de configuration, où la réaction de réticulation a lieu entre CC libres et des protéines en solution à 4 ou l&#39;présentement décrit &quot;sur-talon&quot; de configuration (figure 1B) fonctionne mieux. En général, la méthode doit également être compatible avec n&#39;importe quel état de la protéine d&#39;isotopes stables ou l&#39;art technologie d&#39;étiquetage peptide, ou une évaluation de l&#39;échantillon capture par électrophorèse en gel 2D. Dans le &quot;off-talon&quot; de configuration, il est également possible de capturer des protéines dans des cellules entières (résultats non publiés). Par ailleurs, le médicament ou le site cofacteur liaison d&#39;une protéine peut être décrite par la détermination de la position de réticulation de près par le CC dans la séquence protéique par MS séquençage peptidique. Le mode de fixation d&#39;une petite molécule d&#39;une protéine peut être explorée en utilisant différentes cpositions de fixation CHIMIQUE à la fonction de sélectivité et de différentes longueurs de liaison. Comme indiqué dans la présente étude, les partenaires de la protéine lie également des protéines traitées par la fonction de sélectivité (RplK comme substrat du PRMA) ou inconnus petites interactions protéine molécule (HSA au glnA) peuvent être identifiés. Résumé, la caractéristique supplémentaire de la CCS, la réactivité de photo-réticulation, permet à l&#39;isolement et l&#39;identification de protéines de faible abondance ou de familles de protéines fonctionnelles à partir de mélanges complexes de protéines avec une sensibilité élevée et fournit aux scientifiques un outil supplémentaire pour l&#39;étude de petites molécules - les interactions entre protéines .

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<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>
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<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>
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<td>caproMag&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
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profiling of methyltransferases and other s-adenosyl-l-homocysteine-binding proteins by capture compound mass spectrometry (ccms)

Il ya une variété d&#39;approches pour réduire la complexité du protéome sur la base de petites molécules fonctionnelles interactions protéine-telles que la chromatographie d&#39;affinité ou d&#39;une protéine de profilage Activity Based 2. Composés de capture trifonctionnelle (CC, figure 1A) 3 sont à la base d&#39;une approche générique, dans lequel l&#39;équilibre initial axé sur l&#39;interaction entre une sonde petite molécule (la fonction de sélectivité, ici S-adénosyl-L-homocystéine, HSA, figure 1A) et les protéines cibles est irréversiblement fixée sur la photo-réticulation entre une fonction de réactivité photo activables indépendante (ici un phénylazide) du CC et la surface des protéines cibles. La fonction de tri (ici biotine) sert à isoler le CC - conjugués de protéines à partir de mélanges complexes biologiques à l&#39;aide d&#39;une phase solide (ici des billes de streptavidine magnétique). Deux configurations de ces expériences sont possibles: &quot;off-talon&quot; 4 ou le décrit présentement «sur-talon&quot; de configuration (figure 1B). La fonction de sélectivité peut être pratiquement n&#39;importe quelle petite molécule d&#39;intérêt (substrats, inhibiteurs, des molécules de médicaments). S-adénosyl-L-méthionine (SAM, figure 1A) est probablement, la deuxième à l&#39;ATP, le cofacteur le plus largement utilisé dans la nature 5, 6. Il est utilisé comme donneur de groupe méthyle majeur dans tous les organismes vivants avec la réaction chimique catalysée par SAM-dépendante méthyltransférases (MTases), qui méthylate 7 de l&#39;ADN, l&#39;ARN 8, 9 protéines, ou des petites molécules 10. Étant donné le rôle crucial des réactions de méthylation dans divers scénarios physiologiques (la régulation des gènes, l&#39;épigénétique, le métabolisme), le profilage des MTases peut être appelé à devenir d&#39;une importance similaire dans la protéomique fonctionnelle le profilage de kinases. Des outils analytiques pour leur profilage, cependant, n&#39;ont pas été disponibles. Nous avons récemment introduit un CC avec HSA en tant que groupe de sélectivité pour combler ce fossé technologique (figure 1A). HSA, le produit de la SAM après le transfert de méthyle, est un inhibiteur général des produits connus MTase 11. Pour cette raison et parce que le naturel cofacteur SAM est utilisé par d&#39;autres enzymes de transférer d&#39;autres parties du cofacteur ou initier des réactions radicalaires ainsi qu&#39;en raison de son instabilité chimique 12, SEP est une fonction de sélectivité idéale pour un CC de cibler MTases. Nous rapportons ici l&#39;utilité de la SAH-CC et CCMS par MTases profilage et d&#39;autres protéines liant l&#39;HSA de la souche DH5a d&#39;Escherichia coli (E. coli), l&#39;un des mieux caractérisés les procaryotes, qui a servi de modèle préféré organisme dans d&#39;innombrables études biochimiques, biologiques et biotechnologiques. Photo-activé réticulation améliore le rendement et la sensibilité de l&#39;expérience, et la spécificité peuvent être facilement testés dans des expériences de compétition en utilisant un excès du libre HSA.

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

Composés de capture sont trifonctionnel petites molécules afin de réduire la complexité du protéome par fonctionnels réversibles petite molécule de protéine d&#39;interaction suivie par la photo-réticulation et de purification. Ici, nous utilisons un composé de capture avec des<em&gt; S</em&gt;-Adénosyl-<sub&gt; L</sub&gt;-Homocystéine-contraignante que la fonction de sélectivité pour isoler méthyltransférases d&#39;un<em&gt; Escherichia coli</em&gt; Cellule entière lysat et les identifier par la SP.

Profilage de la méthyltransférase et autres S-Adénosyl- L-Homocystéine protéines liant par spectrométrie de masse de captation composé (CDSM)

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 vues.  caprotec bioanalytics GmbH. Composés de capture sont trifonctionnel petites molécules afin de réduire la complexité du protéome par fonctionnels réversibles petite molécule de protéine d'interaction suivie par la photo-réticulation et de purification. Ici, nous utilisons un composé de capture avec des S-Adénosyl- L-Homocystéine-contraignante que la fonction de sélectivité pour isoler méthyltransférases d'un Escherichia coli Cellu...

Vidéo: 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).

Préparation des échantillons MALDI: la méthode de la couche ultra mince

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

Recherche

Biologie

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.

Profilage anticorps par immunoprécipitation Systèmes luciférase (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.

L&#39;analyse des complexes protéiques par spectrométrie de masse Grande structurels

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.

Profilage de masse Oligo (OLIMP) de polysaccharides extracellulaires

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.

Pression atmosphérique imagerie moléculaire des tissus biologiques et biofilms par spectrométrie de masse LAESI

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.

Analyse directe des cellules isolées par spectrométrie de masse à la pression atmosphérique

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.

La quantification des protéines à l&#39;aide d&#39;enrichissement d&#39;immunoaffinité peptide couplé à la spectrométrie de masse

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.

Profilage thiol redox du protéome par spectrométrie de masse isotopique de l&#39;étiquetage

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.

Captation composé spectrométrie de masse - un outil puissant pour identifier de nouveaux c-di-GMP protéines effectrices

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.

Étiquetage spécifique de la séquence des acides nucléiques et des protéines avec Methyltransferases et le cofacteur Analogues

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