Cette recherche a été soutenue par le fonds de démarrage de l&#39;Université Simon Fraser.

1. Récolte membranes <ol><li> Ajouter un tampon hypotonique (10 mM Tris-HCl, 10 mM de KCl, 1,5 mM MgCl 2, pH 8,0) avec un cocktail inhibiteur de protease sur un culot de cellules (~ 10 8 cellules) et incuber pendant 15 à 30 min sur de la glace. Lyse des cellules par l&#39;échantillon au travers des aiguilles de seringues (5-10 passes) ou l&#39;homogénéisation de l&#39;échantillon par un homogénéisateur Dounce (15-30 coups). Utilisez un hématimètre et coloration au bleu trypan pour vérifier l&#39;efficacité de la lyse. </li><li> Obtenir la fraction microsomale par une centrifugation différentielle du lysat à 3000 gx 15 min puis à 100 000 gx 2 h. La première centrifugation obtient un culot qui est la fraction nucléaire, et la centrifugation subséquente du surnageant génère une deuxième pastille, qui est la fraction microsomale qui contient la membrane plasmique ainsi que des organelles liées à la membrane 8. Le surnageant final est la fraction cytosolique. Stocker les fractions microsomales dans le congélateur à -80 ° C pour de plus amples analysest comme indiqué ci-dessous. </li></ol> 2. Dissoudre, Dénaturer, et Digest protéines membranaires <ol><li> Dissoudre la fraction microsomale dans le tampon de dénaturation (Tris 40 mM, EDTA 10 mM, TCEP 10 mM, 0,5% Rapigest, pH 8) et ensuite incuber la solution à 100 ° C pendant 10 min. </li><li> On refroidit la solution chauffée à température ambiante, ajouter à ultra-haute pureté de l&#39;urée en poudre dans la solution à une concentration finale de 8 M et incuber l&#39;échantillon à 37 ° C pendant 30 min. </li><li> Ajouter la solution mère de l&#39;iodoacétamide à l&#39;échantillon à 15 mM de concentration finale, et incuber la solution à l&#39;obscurité pendant 30 min à température ambiante pour alkyler les thiols libres dans l&#39;échantillon. Ajouter du DTT solution mère de l&#39;échantillon à une concentration finale de 10 mM et incuber la solution pendant encore 10 min à température ambiante pour étancher l&#39;iodoacétamide excessive. </li><li> Diluer la solution obtenue de 10x avec un tampon Tris 40 mM à pH 8, et à la suite de la trypsine à l&#39;échantillon dans un rapport de 01:20 trypsin de la protéine totale. Maintenir la réaction de digestion à 37 ° C un four pendant une nuit pour s&#39;assurer que la réaction est terminée. </li><li> Mettre fin à la digestion par l&#39;acidification de la solution d&#39;échantillon à pH 1 avec HCl, une condition qui décompose également le détergent, à savoir Rapigest. Dégrader la Rapigest à 37 ° C pendant 1 heure, et enlever la précipitation développé par centrifugation. </li><li> Nettoyer le surnageant qui contient les peptides échantillon par une cartouche C-18 d&#39;extraction en phase solide (SPE) et sécher l&#39;échantillon obtenu par speedvac. </li><li> Effectuer une analyse SDS-PAGE des échantillons avant et après digestion à la trypsine afin de confirmer l&#39;efficacité de la digestion. </li></ol> 3. Glycopeptides capture <ol><li> Dissoudre les peptides nettoyés dans le tampon de couplage (acétate de sodium 100 mM, pH 5,5). Ajouter le periodate de sodium dans la solution de peptide à une concentration finale de 10 mM pendant 30 min à l&#39;obscurité, à température ambiante. Cela permettra d&#39;oxyder les groupes cis-diol dans les glycanes en aldéhydes, Qui permettent de coupler les glycanes de la résine à travers la chimie hydrazide. Étancher le periodate excessive par le sulfite de sodium à une concentration finale de 20 mM et à pH 5 pendant 10 min à température ambiante. </li><li> Introduire la résine transformée en dérivé hydrazide dans la solution de peptide à un rapport de 1:4 (solution de résine) pour coupler des glycopeptides à la résine. Incuber la réaction à 37 ° C pendant 1 à 2 jours avec la rotation de bout en bout pour le couplage complet. </li><li> Éliminer les peptides non liés par lavage de la résine à deux reprises avec 1 ml de chacun des éléments suivants: l&#39;eau DI, 1,5 M de NaCl, de méthanol et 80% d&#39;acétonitrile. Enfin, le lavage de la résine avec 3 x 1 ml de 100 mM de NH 4 HCO 3 à pH 8 pour échanger le tampon du système à 100 mM de NH 4 HCO 3. <ol><li> Recueillir le surnageant et les lavages à l&#39;analyse des peptides non liés (en option). </li></ol></li><li> Libérer les N-glycopeptides à partir de la résine par la PNGase F à une incubation pendant une nuit à 37 ° C avec unn bout-à-bout rotation. Recueillir les peptides libérés par centrifugation et un lavage à l&#39;acétonitrile 80%. Sécher la solution obtenue dans l&#39;speedvac pour l&#39;analyse LC-MS. </li></ol> 4. Fractionnement supplémentaire (en option) Pour simplifier encore plus la complexité de l&#39;échantillon, fractionner les N-glycopeptides obtenus. Par exemple, les peptides séchés redissoudre dans 10 mM de formiate de l&#39;ammoniac, un pH de 3 avec 20% d&#39;acétonitrile et en utilisant une forte échangeuse de cations (SCX) de chromatographie pour fractionner les peptides. Sécher l&#39;éluant obtenu, puis analyser les fractions peptidiques obtenues par phase inverse LC-MS 8,17,18. 5. Nettoyage des N-glycopeptides libérés (Facultatif) Si les préoccupations augmentent pour la contamination potentielle des peptides, dissoudre les peptides secs dans de l&#39;acide formique à 0,1% et d&#39;utiliser une colonne MCX SPE pour nettoyer en outre les peptides avant l&#39;analyse de phase inverse LC-MS. <strong&gt; Remarque: les paramètres de la base de données Searching. Lors de la coupure sélective de N-glycopeptides au large de la résine, PNGase F convertit l&#39;asparagine N-glycane lié à un acide aspartique. Par conséquent, il existe un décalage 0,9840 Da masse des N-glycopeptides libérées. Pour identifier avec précision ces peptides, cette modification doit être ajouté à des paramètres de recherche ainsi que des modifications communes telles que la carbamidomethylation de la cystéine et de l&#39;oxydation de la méthionine.

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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Ici, nous introduisons une stratégie glycopeptides capture pour le profilage des protéines de surface cellulaire. Le procédé peut être appliqué pour étudier les protéines sécrétées, tels que ceux dans le sang, ainsi que dans d&#39;autres liquides corporels ou dans des milieux de culture cellulaire. Le succès de la méthode repose sur la digestion complète d&#39;échantillons; Par conséquent, une SDS-PAGE, la caractérisation de l&#39;efficacité de la digestion est nécessaire,...

les protéines de surface cellulaire interagissent avec l&#39;environnement extracellulaire et transmettent des signaux à partir de l&#39;extérieur vers l&#39;intérieur d&#39;une cellule. Ainsi, ces protéines, comprenant des protéines de la matrice extracellulaire, jouent un rôle essentiel dans tous les aspects de la biologie cellulaire et la physiologie allant de la prolifération, la croissance, la migration, la différenciation au vieillissement et ainsi de suite. les protéines de surface fonctionnent en interaction avec d&#39;autres cellules, des protéines et des petites molécules 3.1. La caractérisation moléculaire des protéines de surface cellulaire est d&#39;un grand intérêt non seulement pour les biologistes, mais aussi pour les sociétés pharmaceutiques, comme plus de 60% ​​des médicaments sont destinées aux protéines de surface cellulaire 4. Spectrométrie de masse en tandem (MS), avec sa sensibilité supérieure, la précision et débit pour l&#39;identification des protéines et des peptides, a été un outil puissant pour les études protéomiques mondiaux 5,6. Pourtant, les protéines de surface posent des défis importants à la protéomique MS, commela plupart des protéines de surface existent en faibles quantités et avec des modifications lourdes. Les régions transmembranaires des protéines de surface rendent hydrophobe; ce qui est particulièrement le cas pour les protéines transmembranaires multipass. Il est donc difficile de dissoudre les protéines membranaires dans des solutions aqueuses, sans l&#39;aide d&#39;un détergent; toutefois, l&#39;utilisation de détergents supprime généralement la performance de HPLC et MS 1,7,8 dans l&#39;identification des protéines. Par conséquent, les protéines membranaires ont été mal caractérisé en protéomique basées directs LC-MS. La glycosylation est l&#39;une des modifications post-traductionnelles plus importantes et abondantes qui ont lieu dans les protéines de surface cellulaire 9. L&#39;énorme complexité et l&#39;hétérogénéité des glycanes entravent MS signal de peptides 10. Néanmoins, plusieurs procédés protéomiques ont utilisé cette modification unique pour enrichir les protéines de surface et d&#39;éliminer les fragments de sucre à partir de protéines avant l&#39;analyse LC-MS. Ces procédés comprennent basé lectine capture par affinité 11 et à base d&#39;acide capture chimique ou borique base hydrazide-12 ainsi que des séparations chromatographiques hydrophiles 8,13. La suppression des glycanes transforme les protéines membranaires des protéines réguliers et radicalement simplifie la caractérisation MS. Parce que la glycosylation a également lieu dans les protéines sécrétées qui ont une forte solubilité contrairement aux protéines membranaires, de nombreuses méthodes glycoproteomic sont optimisés pour les protéines solubles, et ont tendance à avoir plus faible sélectivité des glycopeptides et sensibilité lorsqu&#39;il est déployé aux protéines membranaires 8,14. D&#39;autres procédés existent également à enrichir, en particulier, des protéines de surface cellulaire, tels que ceux utilisant une ultracentrifugation 15 et stratégies de marquage 16. Une comparaison détaillée entre notre méthode et d&#39;autres méthodes existantes pour caractériser les protéines membranaires a été menée récemment 17, et les résultats ont indiqué que notre méthode peut effectuer aussi bien, si pasmieux, que toutes les méthodes de protéomique de la membrane par rapport, mais avec simplicité supérieur. Pour aider les chercheurs utilisent cette méthode, nous détaillons ici un protocole général. Cette méthode intègre plusieurs avantages de stratégies de glycoprotéomique existantes et est conçu spécifiquement pour les glycoprotéines membranaires, encore la méthode fonctionne aussi bien pour des protéines sécrétées. Les caractéristiques de ce procédé comprennent: 1) une solubilisation complète de protéines membranaires, 2) l&#39;enrichissement du glycopeptides au lieu de glycoprotéines pour éliminer l&#39;encombrement stérique potentiel lors de l&#39;utilisation d&#39;un substrat solide de capture, 3) l&#39;utilisation de la chimie de l&#39;hydrazide pour former des liaisons covalentes entre glycopeptides et le substrat de capture, telles que des glycopeptides liés peuvent tolérer des lavages strictes pour haute glycoselectivity, et 4) la capacité d&#39;effectuer la procédure de capture dans l&#39;ensemble de tube pour une perte réduite de l&#39;échantillon et de la durée de la procédure raccourcie. Après l&#39;application de cette méthode à l&#39;étude d&#39;une variableciété d&#39;échantillons biologiques, y compris les cellules et les tissus, nous avons observé une sélectivité élevée (&gt; 90%) à des glycoprotéines 8,17,18.

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646563</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodoacetamide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A3221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rapigest SF</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186001860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium periodate</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">311448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNGase F</td>
			<td style="width:199px;">New England Biolabs</td>
			<td style="width:119px;">P0704S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Affi-Gel Hz Hydrazide gel</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">153-6047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:199px;">Worthington Biomedical</td>
			<td style="width:119px;">LS02115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sep-Pak C-18 cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">WAT054955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX cartridge</td>
			<td style="width:199px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor coctail</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:199px;">Amersco</td>
			<td style="width:119px;">568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium sulphite</td>
			<td style="width:199px;">Caledon</td>
			<td style="width:119px;">8360-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Invertase</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">I0408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alpha-1 trypsin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ribonulease B</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">R7884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Avidin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ovalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">C0755</td>
			<td></td>
		</tr>
	</tbody>
</table>

glycopeptide capture for cell surface proteomics

les protéines de surface cellulaire, y compris les protéines de la matrice extracellulaire, de participer à tous les processus et les fonctions cellulaires importantes, telles que la croissance, la différenciation et la prolifération. Une caractérisation complète de ces protéines fournit de l&#39;information riche pour la découverte de biomarqueurs, l&#39;identification du type de cellule, et la sélection cible thérapeutique, ainsi que d&#39;aider à faire progresser notre compréhension de la biologie cellulaire et physiologie. protéines de surface, toutefois, posent des défis analytiques importantes, en raison de leur nature faible abondance, hydrophobie élevée, et des modifications post-traductionnelles lourds. Profitant de la glycosylation répandue sur les protéines de surface, nous présentons ici une approche glycopeptides capture à haut débit qui intègre les avantages de plusieurs moyens N-glycoprotéomique existants. Notre méthode peut enrichir les glycopeptides dérivés des protéines de surface et de retirer leurs glycanes pour la protéomique faciles à l&#39;aide de LC-MS. La N-glycoproteome résolu comprend l&#39;information de l&#39;identité de la protéine et la quantité ainsi que de leurs sites de glycosylation. Cette méthode a été appliquée à une série d&#39;études dans des domaines tels que le cancer, les cellules souches, et la toxicité des médicaments. La limitation de la méthode réside dans la faible abondance des protéines membranaires de surface, de telle sorte qu&#39;une quantité relativement importante d&#39;échantillons est nécessaire pour cette analyse par rapport à des études ont porté sur des protéines cytosoliques.

Un organigramme représentant la procédure expérimentale est résumée sur la figure 1. L&#39;étiquetage et d&#39;autres étapes de fractionnement sont facultatifs et les détails sont décrits dans une publication récente 18. Une autre option est d&#39;analyser les peptides non modifiés, qui ne réagissent pas avec la résine. Les avantages de l&#39;analyse des peptides non modifiés comprennent l&#39;identification potentielle des peptides et des protéines non glycosylées, telles que...

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Glycopeptide Capture for Cell Surface Proteomics

les protéines de surface cellulaire sont biologiquement importantes et largement glycosylée. Nous présentons ici une approche glycopeptides capture pour solubiliser, enrichir et deglycosylate ces protéines pour les analyses protéomiques faciles à base LC-MS.

Glycopeptides Capture pour protéomique de surface cellulaire

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

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11.2K vues.  Simon Fraser University.  les protéines de surface cellulaire sont biologiquement importantes et largement glycosylée. Nous présentons ici une approche glycopeptides capture pour solubiliser, enrichir et deglycosylate ces protéines pour les analyses protéomiques faciles à base LC-MS. 

Vidéo: Glycopeptide Capture for Cell Surface Proteomics

Cell Capture Using a Microfluidic Device

Capturez cellule à l&#39;aide d&#39;un dispositif microfluidique

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Biologie

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

L&#39;avidité d&#39;un dépistage interaction extracellulaire (AVEXIS) pour la détection évolutive de faible affinité extracellulaires interactions récepteur-ligand

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Marquage métabolique et la membrane de fractionnement pour comparative Analyse protéomique de<em&gt; Arabidopsis thaliana</em&gt; Cultures Suspension cellulaires

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Protéomique Préparation de l&#39;échantillon de formol et inclus en paraffine fixe tissus

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

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 ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

Non enzymatique, sans sérum de culture de tissus de lésions mammaires pré-invasives pour la génération spontanée de mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

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

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

Selected Reaction Monitoring spectrométrie de masse pour Absolute quantification des protéines

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

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 ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Répandre surface et Immunocoloration des chromosomes de levure

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Analyse de surface cellulaire Adhérence remodelage en réponse à la tension mécanique utilisant des billes magnétiques

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...