We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1. Préparation Lysate <ol><li> Cultivez P. cellules aeruginosa dans LB à la DO souhaitée. REMARQUE: Pour obtenir des conseils: utiliser ≈ 100 ml de culture / échantillon pour cultures en phase stationnaire et ≈ 500 ml cultur / échantillon pour les cultures en phase logarithmique (DO 600 nm = 0,5). </li><li> Pellet par centrifugation pendant 20 min à 5000 x g. </li><li> Remettre en suspension de 0,5 à 1 g de culot dans 1 ml de tampon de lyse (mM MES 6,7 mM HEPES 6,7 mM, NaCl 200 mM KAC 6,7, DDT 1 mM, pH 7,5) et ajouter inhibiteur de la protéase (mini complète, sans EDTA) ainsi que DNaseI. </li><li> Lyse des cellules par trois passages à travers une cellule de pression française, à 20 000 psi (voir liste des matières). </li><li> Ultra-centrifugeuse de la lysat cellulaire à 100 000 x g pendant 1 h à 4 ° C. </li><li> Enregistrez le surnageant (passez à l&#39;étape 2). </li><li> Laver le culot avec 1 ml de tampon de lyse 1x par pipetage de haut en bas. </li><li> Ultracentrifugeuse à 100 000 x g pendant 1 heure à 4 ° C. </li><li> Flash geler le culot dansazote liquide et conserver à -20 ° C jusqu&#39;à utilisé pour la capture des protéines membranaires (voir l&#39;étape 3). </li></ol> 2. Suppression des nucléotides libres c-di-GMP et les autres (fraction soluble uniquement) <ol><li> Laver une colonne PD10 de dessalage (voir la liste des matériaux) avec 10 ml de tampon de lyse froid. </li><li> Verser le surnageant (≈ 1 ml) sur la PD10 afin d&#39;éliminer les nucléotides. </li><li> Eluer avec 4 ml de tampon de lyse froid (500 étapes pi). </li><li> Sélectionnez les fractions les plus concentrées comme déterminé par dosage de Bradford, et mettre en commun leur. </li></ol> 3. Pellet et resuspension solubilisation (Fraction membrane seulement) <ol><li> Reprendre le culot dans 500 à 1000 pi de tampon 1x de capture (sans détergent) (voir le tableau 1). NOTE: Le culot est difficile de remettre en suspension. Il est conseillé à la première pipette de haut en bas avec une pipette pour remettre en suspension à peu près le culot, puis d&#39;utiliser une seringue 27 G pour bien homogénéiser la solution. </li><li> Ajouter 1% (p / v) de n-dodécyl-β-D-maltopyranoside (DDM). </li><li> Incuber à 4 ° C pendant au moins 2 heures (ou O / N) sur une roue rotative. </li><li> Ultracentrifugeuse à 100 000 x g pendant 1 heure à 4 ° C. </li><li> Récolter le surnageant. </li></ol> 4. Concentration en protéines Mesure <ol><li> Mesurer la concentration en protéine par dosage de Bradford (par dosage BCA pour la fraction de membrane). </li><li> Régler la concentration totale en protéine de 10 mg / ml. </li></ol> 5. capture <ol><li> Mélanger 300 ug de protéine avec 1 mM de PIB, GTP, ATP, CTP, et 20 ul de tampon de capture 5x (100 mM de HEPES, 250 mM de KAc, MgAc 50 mM, 5% de glycerol, pH 7,5) (tableau 1). Ajuster le volume de réaction de 100 ul avec du H 2 O. REMARQUE: le volume total inclut le volume de la CDG-CC et c-di-GMP en cas de contrôle de la concurrence (voir l&#39;étape 5.3 et le tableau 2). REMARQUE: Les expériences ont été effectuées dans 200 &amp;bandes l 12 tubes PCR (Thermo Scientific); # 181. REMARQUE: pour la fraction membranaire, assurez-vous de toujours garder la concentration de DDM-dessus de la concentration micellaire critique (0,01% p / v) jusqu&#39;à ce que l&#39;étape de solubilisation de protéines dans 8 M d&#39;urée (étape 7.1). </li><li> Incuber à 4 ° C pendant 30 min sur une roue rotative. </li><li> Ajouter 10 uM CDG-CC (concentration finale). REMARQUE: inclure un contrôle sans CDG-CC (dénommé «contrôle de perles»), et un contrôle additionné de 1 mM c-di-GMP (&quot;de contrôle de la concurrence&quot;) (voir le tableau 2). NOTE: la concentration CDG-CC peut être réglée de 1 à 10 uM. </li><li> Incuber à 4 ° C pendant au moins 2 heures (O / N pour la fraction de membrane) sur une roue rotative, dans l&#39;obscurité. </li><li> Après une courte rotation, réticulation par l&#39;activation de la partie réactive de la CDG-CC avec une lumière UV pendant 4 min, en utilisant un CaproBox (voir la liste des matériaux, λ = 310 nm, Irradiance ≥10 mW / cm², la distance de la source = 2 cm). REMARQUE: retirer le couvercle des bandes croisées de liaison avant. </li><li> Ajouter 25 ul 5x tampon de lavage (5 M de NaCl, Tris 250 mM, pH 7,5) et 50 pi de billes magnétiques de streptavidine ainsi remis en suspension, homogénéiser délicatement. </li><li> Incuber à 4 ° C pendant 1 heure sur une roue rotative. </li></ol> 6. étapes de lavage NOTE: (Magnet: voir liste des matières). Commencez avec une capture des billes magnétiques dans le couvercle de la bande PCR, avec l&#39;aimant. Puis remplacer la bande PCR par un nouveau contenant la solution de lavage suivant. Retirer l&#39;aimant et remettre en suspension les perles, et incuber 2 min. Isoler et remplacer le couvercle par un couvercle frais. <ol><li> Les étapes de lavage (fraction soluble uniquement) <ol><li> Laver 6 fois dans 200 tampon de lavage ul 1x. </li><li> Laver une fois dans 200 pi qualité HPLC H 2 O. </li><li> Laver 6 fois dans 200 pi 80% d&#39;acétonitrile. </li><li> Laver deux fois dans 200 pi qualité HPLC H 2 O. </li></ol></li><li> Les étapes de lavage (membrane partie seulement) <ol><li> Laver 5 fois dans 200 pi de tampon de lavage 1x + 0,1% DDM. </li><li> Laver deux fois dans 200 pi de tampon de lavage 1x + 0,05% DDM. </li><li> Laver une fois dans 200 pi de tampon de lavage 1x + 0,025% DDM. </li><li> Laver une fois dans 200 pi de tampon de lavage 1x + 0,0125% DDM. </li><li> Lavez 3 fois dans 200 pi mM ABC 100 (bicarbonate d&#39;ammonium, NH 4 CO 3) + 2 M d&#39;urée. </li></ol></li></ol> 7. MS Préparation de l&#39;échantillon <ol><li> Resuspendre les billes (directement dans le couvercle) dans 20 ul ABC 100 mM (100 mM ABC + 8 M d&#39;urée pour la fraction de membrane) et transférer dans des tubes de 1,5 ml. </li><li> Fraction membranaire seulement: incuber à 60 ° C pendant 5 min, sous agitation à 500 tours par minute. </li><li> Ajouter 0,5 ul mM PTCE 200 (tris (2-carboxyéthyl) phosphine) et incuber à 60 ° C pendant 1 heure, en agitant à 500 rpm. Refroidir à 25 ° C. </li><li> Ajouter 0,5 ul de 400 mM fraîchement préparée iodoacétamide et incuber à 25 ° C pendant 30 min, en secouant uneT 500 tours par minute et dans l&#39;obscurité. </li><li> Ajouter 0,5 pi de 0,5 M de N-acétyl-cystéine et incuber à 25 ° C pendant 10 min, sous agitation à 500 tours par minute. </li><li> Fraction membranaire uniquement: ajouter 1 pl Lys-C et incuber à 37 ° C, O / N. </li><li> Ajouter 2 pg de trypsine et incuber O / N à 37 ° C, en agitant à 500 rpm (enveloppement dans Parafilm pour éviter le dessèchement). NOTE: les échantillons peuvent être conservés à -20 ° C à ce stade. REMARQUE: fraction membrane uniquement: ajouter 100 mM ABC pour ajuster la concentration de l&#39;urée à &lt;2 M avant l&#39;addition de trypsine. </li><li> Brièvement centrifuger les tubes et de recueillir des perles avec l&#39;aimant. </li><li> Transférer le surnageant dans un nouveau tube de 1,5 ml (répéter cette étape si les perles sont de gauche). </li><li> Ajouter 5 ul de 5% de TFA (acide trifluoroacétique) + 1 ul de HCl 2 M (+ HCl 5 M ul 15 ul de 5% de TFA 2 pour la fraction de membrane). </li><li> Condition colonnes C18 MicroSpin (Le Groupe de Nest, MA, USA) avec 150 ul acétonitrile (spin 20 secondes à 2400 tours par minute). </li><li> Equilibrate les colonnes C18 deux fois avec 150 ul de 0,1% de TFA (spin 20 sec, 2400 rpm). </li><li> Chargez l&#39;échantillon et de spin 2 min, 2000 rpm. </li><li> Recharger l&#39;écoulement sur la colonne et répéter l&#39;étape de filage (2 min, 2000 rpm). </li><li> Laver trois fois avec 150 ul de 0,1% de TFA, 5% d&#39;acétonitrile (20 sec, 2400 rpm). </li><li> Prendre un nouveau tube et on élue deux fois avec 150 ul de 0,1% de TFA, 50% d&#39;acétonitrile (2 min, 2000 rpm). </li><li> Sécher les peptides dans un speed-vac. </li><li> Remettre en suspension dans 40 ul de 98% de H 2 O, 2% d&#39;acétonitrile, d&#39;acide formique à 0,15%. </li><li> Soniquer 20 sec (cycle d&#39;impulsion 0,5, amplitude de 100%; voir la liste des matériaux) et centrifuger 5 sec, 12000 rpm (centrifugeuse de paillasse). Vortex 10 sec, et centrifuger 5 secondes, 12 000 rpm. Transférer dans un flacon de HPLC pour l&#39;analyse LC-MS / MS. </li><li> Congeler à -20 ° C. NOTE: les échantillons peuvent être conservés à -20 ° C à ce stade. </li></ol> 8. LC-MS / MS Analysis <ol><li> Exécutez un nano-LC (systèmes nano-LC) equipped avec une colonne de RP-HPLC (75 pm x 37 cm) garnie de résine C18 (Magic C18 AQ 3 um) en utilisant un gradient linéaire de 95% de solvant A (acide 0,15% de l&#39;acide formique, 2% d&#39;acétonitrile) et 5% de solvant B ( 98% d&#39;acétonitrile, d&#39;acide formique à 0,15%) à 35% de solvant B sur 60 min à un débit de 0,2 ul / min. </li><li> Analyser les peptides par LC-MS / MS (double-pression LTQ Velos Orbitrap spectromètre de masse, relié à une source d&#39;ions à électronébulisation). REMARQUE: Le mode d&#39;acquisition de données a été créée pour obtenir une haute résolution MS SCAN dans la partie FT du spectromètre de masse à une résolution de 60000 FWHM suivie par des analyses MS / MS dans le piège à ions linéaire des 20 ions les plus intenses. Pour augmenter l&#39;efficacité de tentatives MS / MS, la projection modus chargée de l&#39;Etat a été activé à exclure non affecté et charge individuellement ions. Collusion dissociation induite a été déclenchée lorsque le précurseur dépassé les 100 chefs d&#39;ions. La durée d&#39;exclusion dynamique a été réglée à 30 sec. Le temps d&#39;accumulation d&#39;ions a été réglée à 300 ms (MS) et 50msec (MS / MS). </li></ol> 9. Base de données Recherche <ol><li> Télécharger le P. aeruginosa NCBI-base de données via la page d&#39;accueil du NCBI ( <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ). </li><li> Convertir les spectres MS brut en fichiers génériques de mascotte (mgf) en utilisant l&#39;outil de conversion MassMatrix ( <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a> ). </li><li> Chercher dans ce fichier mgf utilisant MASCOT version 2.3 contre le P. aeruginosa NCBI-base de données contenant de l&#39;avant et les entrées de protéines reverse-leurres. </li><li> Effectuer une in silico la digestion de la trypsine après lysine et l&#39;arginine (moins suivi par la proline) tolérer manqué deux clivages dans les peptides tryptiques pleinement. </li><li> Définissez les paramètres de recherche de base de données pour permettre methionines oxydé (15,99491 Da) que des modifications variables et carboxyamidomethylation (57,021464 Da) des résidus cystéine que la modification fixe. Pour MASCOT nous scruteing scans haute résolution, définir la tolérance de masse de précurseur de 15 ppm et définir la tolérance de masse de fragment à 0,6 Da. Enfin, réglez la protéine FDR à 1%. </li><li> Importez les recherches mascotte de P. expériences aeruginosa CDSM dans Échafaudages (Proteomesoftware, Version 3), définissez les paramètres pour obtenir une protéine FDR près de 1%, et extraire les comtes spectrale totale. </li><li> Pour les résultats représentatifs présentés dans cet article, nous avons utilisé un test t apparié pour comparer l&#39;expérience avec le contrôle de la concurrence, et seulement considéré succès avec une valeur de p inférieure à 0,1, et un ratio de comptage spectrale supérieure à 2 (expérimenter compte spectrales / contrôle de la concurrence spectrale chiffres). </li><li> Les données peuvent être exportées dans ne importe quel tableur pour une analyse ultérieure. </li></ol> 10. Quantification Sans étiquette <ol><li> Importez les fichiers bruts dans le logiciel ProGenesis LC-MS (dynamique non linéaire, la version 4.0). </li><li> Effectuez un alignement LC-MS et détection de caractéristiques de Setti par défautNGS. </li><li> Exporter les données au format mgf de ProGenesis LC-MS. </li><li> Rechercher les spectres MS / MS en utilisant la mascotte contre le NCBI P. base de données contenant aeruginosa avant et entrées de protéines reverse-leurres. </li><li> Importez les résultats de la recherche de base de données dans ProGenesis LC-MS et cartographier les peptides identifications aux fonctionnalités MS1. </li><li> Pour l&#39;évaluation des données (calcul des niveaux de signification, plier changement ratios) le script ProteinSQAnalysis de SafeQuant a été utilisée (seuil: valeur de q inférieur à 0,1, le ratio de comptage spectrale supérieure à 2). </li></ol>

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The authors have nothing to disclose.

Une attention particulière devrait être prise à plusieurs étapes du protocole. La concentration en protéine est un paramètre critique avec une concentration de 10 mg / ml étant difficile à atteindre lorsque les cellules sont cultivées dans des conditions de croissance spécifiques (par exemple des biofilms ou des variants petites colonies). Ainsi, la remise en suspension du culot doit être effectuée dans un faible volume de tampon de lyse. Les concentrations de protéines peuvent être diminuées à ...

c-di-GMP est un second messager clé utilisé par la plupart des bactéries de contrôler différents aspects de leur croissance et leur comportement. Par exemple, c-di-GMP régule la progression du cycle cellulaire, la motilité et de l&#39;expression d&#39;exopolysaccharides adhésines de surface et 4/2. Grâce à la coordination de ces procédés c-di-GMP favorise la formation du biofilm, un processus qui est associée à des infections chroniques d&#39;une gamme de 5 bactéries pathogènes. c-di-GMP est synthétisé par des enzymes appelées CYCLASES diguanylate (DGCS) qui abritent un domaine catalytique GGDEF 4. Certains DGCS possèdent un site inhibiteur qui régule négativement l&#39;activité de cyclase lors de la liaison c-di-GMP. La dégradation de c-di-GMP est catalysée par deux classes distinctes des phosphodiestérases (PDE) hébergeant soit un EAL catalytique ou HD-GYP domaine 6,7. La majorité des protéines effectrices connus qui se lient directement c-di-GMP appartiennent à l&#39;une des trois classes de protéines: catalytiqueGGDEF ou EAL domaines allié inactifs et domaines Pilz, petits interrupteurs moléculaires qui subissent des changements conformationnels sur c-di-GMP liaison 8. DGCS, PDE et Pilz protéines sont bien caractérisés et leurs domaines peuvent être prédites in silico de façon relativement sûre. Un intérêt particulier est désormais axée sur l&#39;identification de nouvelles classes d&#39;effecteurs c-di-GMP. Certains effecteurs c-di-GMP avec différents motifs de liaison ont été récemment décrits comme le CRP / FNR Bcam1349 de la famille des protéines dans Burkholderia cenocepacia ou FleQ de régulateur transcriptionnel dans P. aeruginosa 9,10. En outre, riboswitchs-GMP-c-di spécifique ont été récemment identifiés et affichés pour contrôler l&#39;expression génique d&#39;une manière c-di-GMP-dépendante 11. Les motifs c-di-GMP de liaison des différents effecteurs sont seulement mal conservée faire des prédictions bioinformatiques de telles protéines difficile. Pour résoudre ce problème, nous avons développé une méthode biochimique, qui est basé sur l&#39;utilisation d&#39;un spe c-di-GMPcifique de captation composé combinée avec la spectrométrie de masse 1,12,13. Nous avons récemment conçu un roman trivalent c-di-GMP captation composé (CDG-CC, figure 1) 1. Cet échafaudage chimique est composé de: 1) un fragment C-di-GMP utilisée comme appât pour capturer les protéines de liaison à c-di-GMP, 2) un groupe réactif photoactivable-UV utilisé pour réticuler la CDG-CC pour les protéines liées et 3) une biotine d&#39;isoler les protéines capturées en utilisant des billes magnétiques revêtues de streptavidine. Le CDG-CC peut être utilisé pour capturer directement et spécifiquement effecteurs c-di-GMP de mélange complexe de macromolécules que lysats cellulaires. Capturer composé à base de produits chimiques et les approches protéomique base ont déjà été signalés à être applicable à un large éventail d&#39;organismes, par exemple Caulobacter crescentus, Salmonella enterica sérotype typhimurium et P. aeruginosa 1,14. Dans ce document méthodologique,nous fournissons une dans la description de la profondeur de la procédure CCMS en utilisant des extraits de P. aeruginosa titre d&#39;exemple. Cette étude établit CCMS comme un outil puissant et polyvalent pour identifier biochimiquement nouveaux composants impliqués dans la signalisation de petite molécule.

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

Des progrès considérables ont été réalisés au cours de la dernière décennie vers l&#39;identification et la caractérisation des enzymes impliquées dans la synthèse (des cyclases diguanylate) et de dégradation (phosphodiestérases) du second messager c-di-GMP. En revanche, peu d&#39;informations sont disponibles concernant les mécanismes moléculaires et de composants cellulaires à travers laquelle cette molécule de signalisation réglemente un large éventail de processus cellulaires. La plupart des protéines effectrices connus appartiennent à la famille Pilz ou sont dégénéré cyclases ou phosphodiestérases diguanylate qui ont renoncé à la catalyse et ont adopté fonction d&#39;effecteur. Ainsi, afin de mieux définir le réseau c-di-GMP cellulaire dans un large éventail de bactéries méthodes expérimentales sont nécessaires pour identifier et valider de nouveaux effecteurs pour lesquels fiable dans prédictions in silico sûr. Nous avons récemment développé une capture nouveau composé spectrométrie de masse (CCMS) technologie comme un outil puissant pourbiochimiquement identifier et de caractériser les protéines de liaison c-di-GMP. Cette technique a déjà été signalé comme étant applicable à une large gamme d&#39;organismes 1. Ici, nous donnons une description détaillée du protocole que nous utilisons pour sonder ces composants de signalisation. A titre d&#39;exemple, on utilise Pseudomonas aeruginosa, un pathogène opportuniste dans laquelle c-di-GMP joue un rôle essentiel dans la virulence et le contrôle du biofilm. CCMS identifié 74% (38/51) des composants connus ou prévus du réseau c-di-GMP. Cette étude explique la procédure CCMS en détail, et l&#39;établit comme un outil puissant et polyvalent pour identifier de nouveaux composants impliqués dans la signalisation de petite molécule.

Pour identifier de nouveaux effecteurs c-di-GMP dans P. aeruginosa nous avons systématiquement utilisé CCMS pour analyser les fractions solubles et membranaires de P. aeruginosa souche PAO1 d&#39;une culture en phase logarithmique (DO 600 = 0,5). Nous résumons ici et discutons des résultats représentatifs de cette expédition de pêche. Quatre répliques biologiques indépendants ont été utilisés. Pour chaque expérience, deux concentrations CDG-CC différents ont été utilisés (5 ...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

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 ...

capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Research

JoVE Journal

Biology

11.5K vues.  Biozentrum of the University of Basel. 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.

Vidéo: Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

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

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Protéomique pour identifier les protéines interagissant avec P2X2 canaux cationiques ligand-dépendants

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Une évaluation quantitative du lipidome levure en utilisant la spectrométrie de masse électrospray ionisation

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-ubiquitine membrane à base de levure double hybride (MYTH) Système: Un outil puissant pour identifier les interactions entre protéines

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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 ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - Une méthode pour identifier transcriptome à l&#39;échelle des sites de liaison des protéines liant l&#39;ARN

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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 ...

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 ...

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

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amide hydrogène / deutérium Analyse Bourse &amp; MALDI-TOF spectrométrie de masse de PAK2 activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

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 ...