Ce travail a été financé par l&#39;énergie Biosciences Institute octroi OO0G01.

La méthode est illustrée OLIMP sur l&#39;hémicellulose majeurs présents dans les parois cellulaires des plantes dicotylédones, xyloglucane, en utilisant une endoglucanase comme glycosylhydrolase. La méthode est démontrée avec l&#39;ensemble des plants d&#39;Arabidopsis comme source de tissus végétaux. Matériau de la matrice extracellulaire et des enzymes peuvent être remplacés en fonction de l&#39;analyse désirée à l&#39;aide de la même procédure. 1. Paroi cellulaire d&#39;isolation <ol><li> Récolte cinq 5 jours de vieux plants d&#39;Arabidopsis entière ou le montant équivalent de votre matériel désiré et de les transférer dans un tube de réaction de 1.5 ml. Assurez-vous que le matériau est placé dans le fond du tube. </li><li> Ajouter deux boules de métal de 3 mm pour le tube de réaction sur le dessus de l&#39;échantillon et enclenchez-gel dans l&#39;azote liquide. </li><li> Broyer l&#39;échantillon congelé en utilisant un broyeur à billes (2:30 min, 25Hz). </li><li> Ajouter 1ml d&#39;éthanol aqueux à 70% de l&#39;échantillon. Retirez les boules de métal à l&#39;aide d&#39;un aimant. Vortex à fond. </li><li> Centrifuger l&#39;échantillon à 14 000 rpm pendant 10 minutes pour culotter les résidus d&#39;alcool insolubles contenant le matériau de paroi cellulaire. </li><li> Retirer délicatement le surnageant par aspiration. Veillez à ne pas perturber le culot. </li><li> Ajouter 1 ml de chloroforme / méthanol (1:1 v / v). Vortex soigneusement de remettre en suspension le culot. </li><li> Centrifuger l&#39;échantillon à 14 000 rpm pendant 10 min et retirez soigneusement le surnageant par aspiration. Veillez à ne pas perturber le culot. </li><li> Sécher l&#39;échantillon à l&#39;aide d&#39;un concentrateur. </li></ol> 2. Solubilisation d&#39;oligosaccharides <ol><li> Pour la solubilisation du polysaccharide particulier sous forme de son oligosaccharides resuspendre le culot sec dans 25 pi d&#39;un tampon approprié. Pour le formiate d&#39;ammonium 50 mM endoglucanase, pH 4,5, est utilisé. Ajouter 0.2U d&#39;endoglucanase. Vortex de la suspension et liquide de spin bas. </li><li> Incuber l&#39;échantillon pendant 16 heures à 37 ° C dans un incubateur, en agitant à 300rpm. </li><li> Retirez le solvant de la digestion (eau) en utilisant un concentrateur. </li></ol> 3. Analyse MALDI-TOF des oligosaccharides libérés <ol><li> BioRex MSZ 501 perles résine échangeuse de cations sont utilisés pour éliminer les sels et les enzymes de l&#39;échantillon digéré. Conditionner les perles en plaçant une aliquote dans une colonne vide et lavage des billes avec de grandes quantités d&#39;eau. </li><li> Ajouter de 50 à 10 perles BioRex à l&#39;échantillon digéré et séché. Puis ajouter de l&#39;eau 10 ul, pour de petites quantités de matières 5uL d&#39;eau peut être utilisée. Plonger les perles dans la solution par une brève filature le tube. Incuber pendant au moins 10min à température ambiante. </li><li> Spot 2μl matrice (2,5-dihydroxybenzoïque (DHB), 10mg/ml dans l&#39;eau) sur une plaque cible MALDI. Evaporer le solvant sous vide matrice conduisant à cristaux chimiques homogènes matrice. </li><li> 2μl spot de la solution échantillon dessalé au sommet de la matrice de cristaux sur la plaque cible. Si vous voulez apercevoir un grand nombre d&#39;échantillons ne conserve à repérer pour un maximum de 3min. Ce délai garantit que le premier échantillon repéré n&#39;est pas encore sèche. Attendez une 2min supplémentaires pour assurer un mélange intime de la re-dissous matrice et les molécules de l&#39;échantillon d&#39;oligosaccharides dans le dernier échantillon repéré. Puis sécher la plaque cible sous vide. Le mélange échantillon / matrice doit se cristalliser en moins de 1min pour permettre la cristallisation homogène. </li><li> Placer la plaque de mire dans un spectromètre de masse MALDI-TOF. La machine doit être configurée en mode réflectron positive avec une tension d&#39;accélération de 20000V et un retard de 350 nm, la gamme de masse choisie doit être de 500 à 3000 Da (peut changer dépendant des oligomères attendus). Démarrer tir au laser et recueillir 100-200 spectres par échantillon, qui sont compilées pour générer un spectre moyen représentatif (figure 2A). Ions représentant oligosaccharides spécifiques peuvent être identifiés par leur masse au cours de charge (m / z) Le ratio. Les intensités d&#39;ions (hauteur du pic) de tous les ions d&#39;intérêt devrait être ajoutée à 100% résulte de l&#39;abondance relative de chaque oligosaccharide dans l&#39;échantillon (figure 2B). </li></ol> 4. Dans l&#39;analyse in situ OLIMP OLIMP peut également être utilisé directement sur les tissus en omettant toute les étapes de préparation du mur. Comme exemple d&#39;Arabidopsis hypocotyles étiolés comme source de tissus végétaux sont utilisés. Encore une fois, une endoglucanase est utilisé pour déterminer la structure de la xyloglucane hémicelluloses. Enzyme et du matériel tissulaire peut être remplacé en fonction de l&#39;analyse désirée à l&#39;aide de la même procédure. <ol><li> Récolte un semis étiolées et le placer sur une plaque de MALDI cible vide et de laisser le jeune plant sec. </li><li> Ajouter 0.5μl endoglucanase (0.4U/μl, dissous dans formiate d&#39;ammonium 50 mM, pH 4,5) sur le plant séché à la position désirée. Assurez-vous que la chute de l&#39;enzyme touche partiellement la plaque cible. </li><li> Placer la plaque cible MALDI dans un récipient fermé avec humidité saturante pour éviter que la chute de l&#39;enzyme se dessèche. Incuber la plaque dans la chambre pour 16h à 37 ° C. </li><li> Sécher la plaque cible MALDI avec les tissus de la plante et de la chute d&#39;enzymes sous vide. </li><li> Ajouter 0.5μl matrice (DHB; 10mg / l) sur le dessus de chaque spot enzymes séchés. Laisser reposer pendant 2min. </li><li> Sécher la plaque cible MALDI sous vide. </li><li> Analyser les échantillons avec le MALDI-TOF. Placer la plaque de mire avec le tissu et le spot enzyme / matrice (s) dans un spectromètre de masse MALDI-TOF. La machine doit être configurée en mode réflectron positive avec une tension d&#39;accélération de 20000V et un retard de 350 nm, la gamme de masse choisie doit être de 500 à 3000 Da (peut changer dépendant des oligomères attendus). </li><li> Démarrer tir du laser sur les cristaux de matrice / échantillon suivant le tissu. Assurez-vous que vous ne frappez pas le tissu lui-même, que dans un tel cas, le temps de vol des molécules sera éteint. Recueillir autour de 20 à 50 spectres par endroit, qui sont compilées pour générer un spectre moyen représentatif (figure 3). Ions représentant oligosaccharides spécifiques peuvent être identifiés par leur masse au cours de charge (m / z) Le ratio. Les intensités d&#39;ions (hauteur d&#39;ions) peuvent être signalés et tous les ions d&#39;intérêt devrait être ajoutée à 100% résulte de l&#39;abondance relative de chaque oligosaccharides dans l&#39;échantillon. </li></ol> 5. Les résultats représentatifs Un exemple d&#39;une analyse de l&#39;OLIMP xyloglucane hémicelluloses présentes dans plants d&#39;Arabidopsis est montré dans la figure 2. En raison des différences de masse des ions et l&#39;enzyme connue bien caractérisés (endoglucanase) la spécificité des ions peuvent être affectés à des structures spécifiques d&#39;oligosaccharides (figure 2A). Évidemment, les isomères structurels ne peuvent pas être distingués. L&#39;hypothèse de base pour la détermination de l&#39;abondance relative des différents oligosaccharides (figure 2B) est que leur facteur de réponse spectrométrie de masse est très similaire à ceux des oligosaccharides. Comme le montre ici, la quantification OLIMP est hautement reproductible. Toutefois, s&#39;il vous plaît noter que la robustesse de la méthode est très dépendante sur le rapport signal sur bruit des différents ions oligosaccharides. Par exemple la contamination par des sels ou des quantités plus faibles d&#39;oligosaccharides peut diminuer ce ratio. L&#39;analyse OLIMP sur le tissu lui-même (analyse in situ) permet l&#39;étude des zones très petites et définie et implique la préparation des échantillons très peu. Dans l&#39;exemple présenté ici (figure 3) ne qualitatif (mêmes ions), mais des différences quantitatives (variations dans l&#39;intensité des ions) ont été observées entre les pousses et des racines des tissus de la plantule d&#39;Arabidopsis. Permutations des OLIMP-méthode pourrait ainsi conduire à des tissus «imagerie». <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="Figure 1" /> Figure 1. Organigramme de la procédure en utilisant des plants d&#39;Arabidopsis OLIMP entier comme une source végétale. Tout d&#39;abord, le tissu est macéré et matériau de paroi cellulaire est préparée (image modifiée à partir Fujino et al. 10). Puis oligosaccharides d&#39;un polysaccharide mur particulier sont libérés en utilisant une hydrolase spécifique. Enfin, les abondances relatives des oligosaccharides solubles sont déterminées par spectrométrie de masse MALDI-TOF. <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="Figure 2" /> Figure 2. Abondance relative des oligosaccharides xyloglucane dans les semis étiolés d&#39;Arabidopsis tel que déterminé par OLIMP. A) spectre de masse représentant xyloglucane oligosaccharide; chaque ion représente une structure spécifique d&#39;oligosaccharides, les isomères structurels ne peuvent pas être distingués. B) Diagramme à barres correspondant pour la détermination de l&#39;abondance relative d&#39;oligosaccharides. <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="Figure 3" /> Figure 3. Dans l&#39;analyse in situ en utilisant des plants OLIMP étiolés d&#39;Arabidopsis comme exemple. Chaque gouttelette enzyme / digestion (cercles de couleur) peuvent être analysés de manière indépendante et une spectrométrie de masse correspondant peut être obtenu et analysé. <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="Figure 4" /> Figure 4. Spectre OLIMP de l&#39;hémicellulose xylane obtenu par digestion du matériel de feuilles de Miscanthus avec une xylanase (Megazyme).

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+xylan+synthesis+mutants+reveals+new+insight+into+the+mechanisms+of+xylan+synthesis.">Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis.</a> Plant Journal. 52 (6), 1154-1154 (2007).</li><li>Lee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARVUS+gene+is+expressed+in+cells+undergoing+secondary+wall+thickening+and+is+essential+for+glucuronoxylan+biosynthesis.">The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis.</a> Plant Cell Physiol. 48 (12), 1659-1659 (2007).</li><li>Obel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectin+may+hinder+the+unfolding+of+xyloglucan+chains+during+cell+deformation:+implications+of+the+mechanical+performance+of+Arabidopsis+hypocotyls+with+pectin+alterations.">Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations.</a> Molecular Plant . , (2009).</li><li>Cavalier, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+two+Arabidopsis+thaliana+xylosyltransferase+genes+results+in+plants+deficient+in+xyloglucan,+a+major+primary+cell+wall+component.">Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component.</a> Plant Cell. 20 (6), 1519-1519 (2008).</li><li>Hilz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+liquid+chromatography,+capillary+electrophoresis,+and+mass+spectrometry+methods+to+determine+xyloglucan+structures+in+black+currants.">A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants.</a> Journal of Chromatography A. 1133 (1-2), 275-275 (2006).</li><li>Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+mur1+mutation+on+xyloglucans+produced+by+suspension-cultured+Arabidopsis+thaliana+cells.">Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells.</a> Planta. 214 (1), 67-67 (2001).</li></ol>

La méthode présentée ici permet OLIMP une analyse très sensible et rapide des polymères présents dans les matrices extracellulaires. OLIMP combine la libération enzymatique des oligomères avec subséquentes analyse MALDI-TOF. La génération d&#39;un spectre MALDI-TOF prend moins d&#39;une minute, d&#39;où OLIMP est adapté à un large éventail d&#39;applications, notamment à haut débit des études telles que des écrans mutant. OLIMP n&#39;est pas limitée aux polysaccharides végétaux mais peuvent éventuellement être appliquées à une large gamme de polymères, seulement limitée par la disponibilité de certaines enzymes hydrolytiques. Toutefois, une limitation de OLIMP est que l&#39;abondance absolue du polymère ne peut pas être obtenue. Comme mentionné précédemment OLIMP peut être utilisé pour étudier la structure d&#39;une variété de polysaccharides présents dans la matrice extracellulaire de la diversité des espèces. À titre d&#39;exemple, la figure 4 représente un spectre OLIMP de l&#39;hémicellulose majeur dans les graminées, le xylane. Ici, matériau de paroi cellulaire dérivée de l&#39;herbe tempérée Miscanthus a été digéré avec une xylanase.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
<td>&nbsp;</td>		</tbody>
		</table>
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oligo mass profiling (olimp) of extracellular polysaccharides

Le contact direct des cellules à l&#39;environnement est médiatisé dans de nombreux organismes par une matrice extracellulaire. Un aspect commun des matrices extracellulaires est qu&#39;ils contiennent des résidus sucres complexes sous forme de glycoprotéines, des protéoglycanes, et / ou des polysaccharides. Les exemples incluent la matrice extracellulaire des humains et des cellules animales composé principalement de protéines fibrillaires et des protéoglycanes ou les murs polysaccharide à base de cellules de plantes et de champignons, et les protéoglycanes / glycolipide murs à base de cellules de bactéries. Tous ces glycostructures jouent un rôle vital dans la cellule à cellule de communication et de cellule à l&#39;environnement et de signalisation. Un exemple extraordinaire complexité d&#39;une matrice extracellulaire est présente dans les parois des cellules de plantes supérieures. Leur paroi est constituée presque entièrement de sucres, jusqu&#39;à 75% du poids sec, et se compose des biopolymères les plus abondants sur cette planète. Par conséquent, la recherche est menée comment utiliser ces matériaux les mieux comme une ressource renouvelable neutre en carbone pour remplacer les produits pétrochimiques dérivés de combustibles fossiles. Le principal défi pour la conversion de carburant reste à la réticence de murs à la dégradation enzymatique ou chimique due à la glycostructures uniques présents dans cette biocomposites unique. Ici, nous présentons une méthode pour l&#39;analyse rapide et sensible de glycostructures parois cellulaires végétales. Cette méthode de profilage Mass Oligo (OLIMP) est basée la libération enzymatique d&#39;oligosaccharides à partir de matériaux mur de faciliter glycosylhydrolases spécifiques et l&#39;analyse subséquente des mélanges d&#39;oligosaccharides solubilisée à l&#39;aide assistée par matrice désorption / ionisation laser à temps de vol de spectrométrie de masse (MALDI-TOF/MS ) 1 (figure 1). OLIMP nécessite murs de seulement 5000 cellules pour une analyse complète, peut être effectué sur le tissu lui-même deux, et se prêtent à des analyses à haut débit 3. Alors que le montant absolu des oligosaccharides solubilisés ne peut pas être déterminée par OLIMP l&#39;abondance relative des différents ions oligosaccharides peuvent être délimitées à partir des spectres de masse donnant un aperçu sur la substitution modèle du polysaccharide natif présent dans le mur. OLIMP peut être utilisée pour analyser une grande variété de polymères de la paroi, limitée seulement par la disponibilité d&#39;enzymes spécifiques 4. Par exemple, pour l&#39;analyse des polymères présents dans les enzymes de la paroi cellulaire des plantes sont disponibles pour analyser les hémicelluloses xyloglucane utilisant un xyloglucanase 5, 11, 12, 13, xylane utilisant une endo-β 6,7-(1-4)-xylanase , ou pour les polysaccharides pectiques en utilisant une combinaison d&#39;une polygalacturonase et une méthylestérase 8. Par ailleurs, en utilisant les mêmes principes de OLIMP glycosylhydrolase et même des activités glycosyltransférase peuvent être surveillés et déterminée 9.

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

Un moyen rapide est décrite afin de mieux connaître la structure des polysaccharides dans une matrice extracellulaire. La méthode tire parti de la spécificité de glycosylhydrolases et la sensibilité de la spectrométrie de masse permettant d&#39;infimes quantités de matériaux à analyser. Cette technique est adaptable pour être utilisé directement sur le tissu lui-même.

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 ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K vues.  University of California, Berkeley. Un moyen rapide est décrite afin de mieux connaître la structure des polysaccharides dans une matrice extracellulaire. La méthode tire parti de la spécificité de glycosylhydrolases et la sensibilité de la spectrométrie de masse permettant d'infimes quantités de matériaux à analyser. Cette technique est adaptable pour être utilisé directement sur le tissu lui-même.

Vidéo: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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 ...

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

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

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

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Profilage Single Cell transcriptionnelle des cardiomyocytes de souris adultes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profilage Glycan des polymères végétaux de la paroi cellulaire en utilisant Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Analyse de la traduction Initiation Pendant conditions de stress par polysome profilage

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Évaluation de la sélective ARNm traduction dans les cellules de mammifères par polysome profilage

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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.

Enrichissement de matrice extracellulaire de protéines à partir de tissus et la digestion en peptides pour l&#39;analyse de spectrométrie de masse

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Profilage Redox cellulaire utilisant une microscopie haute teneur

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...