Ce travail a été financé par le NCI clinique protéomique Technology Assessment Center (CPTAC) subvention (# U24 CA126476) ainsi que d&#39;une subvention de l&#39;industrie du divertissement de la Fondation (FEI) et le Fonds des femmes FEI recherche sur le cancer au Consortium du cancer du sein découverte de biomarqueurs, et dons généreux de la Fondation Keck, la Fondation des Canaries, et le Paul G. Allen Family Foundation.

Procédure expérimentale: Le dosage nécessite peptides synthétiques et anticorps anti-peptide. Peptides sélectionnés doivent être uniques à la protéine d&#39;intérêt, contenir entre 8 et 22 acides aminés, et n&#39;ont pas connu modifications post-traductionnelles. Résidus méthionine sont généralement évitées et les peptides contenant des acides aminés dibasiques (par exemple, KK, KR, RR) sont indésirables. Pour cette technique, il est courant d&#39;utiliser des peptides isotopes stables étiquetés comme des normes internes, incorporant lourds (13 C et 15 N) des acides aminés marqués au C-terminale du peptide (ie K ou R étiqueté). Le protocole suivant décrit un test conçu pour mesurer l&#39;GDSLAYGLR peptide de la protéine de souris ostéopontine, en utilisant des anticorps anti-peptide obtenu à partir de Epitomics Inc (Burlingame, CA) et des peptides synthétiques de la Nouvelle Angleterre Peptide (Gardner, MA). Le protocole comprend trois étapes principales (figure 1): 1) digestion par la trypsine du mélange de protéines complexes, 2) l&#39;enrichissement des peptides 3) Analyse par spectrométrie de masse. Il sera démontré sur un échantillon de plasma humain dopés avec la protéine ostéopontine souris. 1. Trypsine digestion enzymatique et le nettoyage <ol><li> Dégeler 10 uL de plasma aliquote pur sur glace mouillée. </li><li> Déterminer la concentration en protéines totales par un dosage BCA et centrifuger l&#39;échantillon pour enlever toute matière en suspension. </li><li> Pipeter 10 uL aliquote de son tube de stockage à un mille Deepwell plaque uL et couvrir avec Pierce-mesure film. </li><li> Ajouter 20 ul de l&#39;urée 9M frais / 30mm dithiothréitol (DTT) (dernière concentration en urée 6M / 20mm TNT) à chaque échantillon. </li><li> Incuber pendant 30 minutes à 37 ° C. </li><li> Ajouter 3 pi de produits frais 500 iodoacétamide mM (finale IAM 50mm) à chaque échantillon. </li><li> Incuber pendant 30 minutes dans le noir à température ambiante. </li><li> Ajouter 257 ul de Tris 100 mM (pH 8) (urée dilue à ~ 0,6 M). </li><li> Ajouter 10 uL de solution mère de trypsine (1 ug / uL, car l&#39;enzyme 01h50: ratio de substrat). </li><li> Incuber à 37 ° C pendant une nuit (12-16 heures). </li><li> Ajouter 3 ul d&#39;acide formique pur (concentration finale de 1%). </li><li> Ajouter la norme des isotopes stables (normes multiples sont ajoutés si vous effectuez un test multiplex, ce n&#39;est généralement environ 10 uL contenant 50-100 fmol de peptide isotopiquement marqués standard). </li><li> Laver la plaque cartouche Oasis bien avec 500 ul d&#39;acide formique 0,1% en 80% d&#39;acétonitrile, en rejetant les actions accréditives. Répétez cela 3 fois. </li><li> Equilibrer la plaque de la cartouche en ajoutant 500 ul d&#39;acide formique 0,1% dans l&#39;eau, et jetez le accréditives. Répétez ce 4 fois. </li><li> Charger les échantillons digérer à la plaque de cartouches et d&#39;ajuster le vide de sorte que le débit est très lent. </li><li> Laver avec 500 ul d&#39;acide formique 0,1% dans l&#39;eau, et jetez accréditives. Répétez cela 3 fois. </li><li> Peptides Eluer en ajoutant 2 x 500 ul d&#39;acide formique 0,1% en 80% d&#39;acétonitrile en 1000 ul puits profond plaque (ne jetez pas l&#39;écoulement continu). </li><li> Lyophiliser (ou speedvac) l&#39;éluat à sec. (La lyophilisation est la méthode préférée) </li><li> Reconstituer séchées peptides en ajoutant 50 pl de PBS + 0,03% de CHAPS. </li></ol> 2. L&#39;enrichissement d&#39;immunoaffinité Peptide <ol><li> Transférer l&#39;échantillon à la norme Kingfisher plaques de 96 puits. </li><li> Ajouter 1 antibody ug et 1,5 uL protéine G billes magnétiques recouvertes par cible (il est facultatif pour réticuler l&#39;anticorps sur les billes avant l&#39;addition). Assurez perles sont bien suspendues en secouant ou en vortex. </li><li> Recouvrir la plaque avec du papier. </li><li> Incuber une nuit (12-16 heures) avec douceur tumbling pour assurer perles sont suspendus. </li><li> Centrifuger la plaque à 32 g pendant 5 secondes pour enlever tout liquide de la surface de la feuille. </li><li> Retirez le couvercle en aluminium. </li><li> Lavage et d&#39;élution des perles peut être effectuée manuellement ou de façon automatisée. Cette procédure décrit les étapes automatisées sur une plate-forme Kingfisher. </li><li> Laver les billes deux fois avec 200 ul de PBS + 0,03% de CHAPS (1 minute par lavage). </li><li> Laver les billes une fois avec 200 dilution 1:10 de PBS + 0,03% de CHAPS (1 minute). </li><li> Les peptides sont élués dans 25 ul d&#39;acide acétique à 5% + 0,03% de CHAPS. </li><li> Placer la plaque d&#39;élution sur un aimant et le transfert de l&#39;éluat à une plaque de 96 puits, en prenant soin de ne pas transférer les restes de perles. </li><li> Recouvrir la plaque avec un tapis d&#39;étanchéité. </li><li> La plaque contenant l&#39;éluat est livré à l&#39;spectromètre de masse triple quadrapole pour analyse. </li></ol> 3. L&#39;analyse par suivi de réactions multiples - spectrométrie de masse <ol><li> Transitions pour les MRS / MRM analyse peuvent être sélectionnés et optimisés en infusant une picomole 1 par microlitre de solution de peptide standard dans 30% d&#39;acide formique acetonitrile/0.1% dans le spectromètre de masse à 0,5 ul / min. Une fois de pulvérisation stable est obtenu, de recueillir un spectre MS / MS. </li><li> Les transitions sont sélectionnés à partir du spectre MS / MS en identifiant three ions fragments abondants dans une région du spectre avec peu de bruit. Pour multiplier les-ions chargés des peptides, des fragments Y-ion détecté à m / z&gt; précurseur de m / z sont généralement le meilleur pour le développement de tests SRM / GRM. La figure 2 montre le spectre MS / MS et des ions de transition sélectionné 476,3&gt; 508,3, 579,3 , 692.4 (transitions pour les standards isotopes stables lourds 481,3&gt; 518,3, 589,3, 702,4 non représenté) pour l&#39;GDSLAYGLR peptide. </li><li> Selon la marque et le modèle du spectromètre de masse utilisé, il ya plusieurs paramètres qui peuvent être optimisés pour chaque transition. Ici, nous avons optimisé l&#39;énergie de collision de chaque transition choisie par ramping l&#39;énergie de collision et la surveillance du niveau de signal. Une énergie de collision de 25 a été utilisé pour chaque transition. </li><li> En bref, une configuration typique pour SRM / MRM analyse est la suivante: la phase mobile (A) 0,1% d&#39;acide formique; (B) acétonitrile 90% / 0,1% d&#39;acide formique, 0,3 x 5 mm de colonne piège C18, 75 ID um x 15 cm C18 analytique (Reprosil-Pur C18 AQ, 120 A pores °). Le volume d&#39;injection est de 10 uL et l&#39;échantillon est chargé pendant 5 min à B 3% à un débit total de 3 pi / min et éluée par un gradient linéaire de 3 à 45% B à 300 - 400 Nl / min à 10 min. Conditions sur un QTRAP 4000 (ABSCIEX, Foster City, CA) ont été pulvérisée de tension 2.3kV, température de la source d&#39;ions à 150 ° C, GS1 de 12 ans, et 15 de gaz rideau. </li><li> L&#39;échantillon est analysé par MRS / MRM en injectant 10 uL de l&#39;échantillon. Domaines de pointe sont intégrées pour les peptides légers et lourds utilisant le Skyline programme. 14 Calculer le ratio de l&#39;aire du pic (PAR) de peptide non étiquetés / étiquetés dans chaque échantillon en utilisant la transition la plus abondante qui est exempt de bruit de fond, dans ce cas, la transition y5 (peptide légers 476,3&gt; 579,3, 481,3 lourde standard de peptides&gt; 589.3). </li></ol> 4. Les résultats représentatifs: Mesuré ratios surface du pic (lumière relative aux pointes peptide endogène peptide isotopiquement marqués lourds) fournir une mesure quantitative du peptide cible. La figure 3 montre un chromatogramme par exemple des peptides légers et lourds dans un échantillon SISCAPA enrichi. Notez que les peptides légers et lourds éluer au même moment et de multiples transitions peuvent être surveillés pour chaque peptide pour confirmer l&#39;identité. <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="Figure 1"/> Figure 1. Un aperçu schématique du processus de SISCAPA. Un mélange complexe de protéines sont digérées en peptides. Analytes peptide ciblé (analyte endogène et une pointes marquée par un isotope stable étalon interne) sont enrichis en utilisant des anticorps anti-peptide immobilisé sur la protéine G-particules magnétiques revêtues. Suite à l&#39;isolation, les peptides sont élués ciblés par les particules magnétiques et analysés par spectrométrie de masse pour la quantification par rapport à l&#39;étalon interne. <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="Figure 2"/> Figure 2. Spectre MS / MS de l&#39;GDSLAYGLR peptide montrant la sélection de trois fragments de MRS / MRM transitions. <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="Figure 3"/> Figure 3. Chromatogrammes Exemple montrant le profil de crête d&#39;un analyte peptide lumière (rouge) et la lourde isotopes stables étiquetés standard interne (en bleu). Chromatogrammes de la transition à partir du peptide y5 léger (476,3&gt; 579,3) et lourd des isotopes stables étiquetés standard (481,3&gt; 589,3) sont tracées au fil du temps comme ils éluer à partir du système de chromatographie.

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Aucun conflit d&#39;intérêt déclaré.

L&#39;étape la plus critique dans le protocole tel que décrit est d&#39;assurer les perles restent bien mélangé pendant la période d&#39;incubation. Permettre aux billes de se déposer au fond du puits / flacon se traduira par une variabilité accrue. Il est également important de spin-down tout liquide qui peut rester sur le haut du puits / flacon après la période d&#39;incubation. Digestion par la trypsine reproductible est également critique. La procédure pour la digestion décrit ici a été largement utilisée dans notre laboratoire en collaboration avec SISCAPA; cependant, il est susceptible de méthodes alternatives de la digestion pourrait être optimisée pour un ensemble donné de protéines cibles 15 élution de l&#39;anticorps libre ne pas interférer avec la détection de l&#39;. peptide, probablement parce que l&#39;anticorps est exclu de la colonne ou élue au plus tard les peptides, mais les anticorps peuvent être réticulés aux billes protéines G avant l&#39;enrichissement d&#39;affinité dans des expériences importantes ou si anticorps libre devient un problème. Nous avons également jugé utile de placer un aimant en dessous de la plaque d&#39;échantillon sur le passeur d&#39;échantillons ainsi que le lavage de la colonne piège dans le sens inverse du flux (par rapport au chargement) pour éliminer toutes les billes ou des particules résiduelles. En plus de l&#39;approche décrite par billes magnétiques, la technique peut également être adapté à un format de colonne (ie la chromatographie d&#39;affinité). 12, 16 Une fois que l&#39;utilisateur est à l&#39;aise avec le protocole général, la technique est également prête à plusieurs modifications qui améliorent le dosage global. 11 Premièrement, il est possible d&#39;analyser des analytes multiples dans un seul test en combinant les anticorps dans l&#39;étape d&#39;enrichissement (multiplexage par exemple). Le spectromètre de masse est capable de analysant simultanément un grand nombre d&#39;analytes. Deuxièmement, l&#39;augmentation du volume de l&#39;échantillon original améliore la sensibilité du dosage.

<table border="1"><tbody><tr><th> Nom de la matière </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> 1000ul assiettes creuses et de 96 puits </td><td> Eppendorf </td><td> 951032786 </td></tr><tr><td> Oasis HLB 1 cc cartouches plaque, Sep-pak, 40 mg plaque de 96 puits </td><td> Eaux </td><td> 186003966 </td></tr><tr><td> Kingfisher plaque de 96 puits </td><td> Thermo Fisher Scientific </td><td> 97002540 </td></tr><tr><td> Effacer 96 puits, la plaque murale blanche </td><td> Bio-Rad </td><td> HSP9601 </td></tr><tr><td> Fleuret couverture pour 96 puits </td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> Tapis d&#39;étanchéité Axymat pour 96 puits </td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> X Pierce cinéma </td><td> Sigma-Aldrich </td><td> Z722502 </td></tr><tr><td> Kingfisher processeur bille magnétique avec tête magnétique PCR </td><td> Thermo Fisher Scientific </td><td> HSP 9601 </td></tr></tbody></table> Tableau 1: Matériaux <table border="1"><tbody><tr><th> Nom de la matière </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> L&#39;urée </td><td> Sigma-Aldrich </td><td> U6031 </td><td></td></tr><tr><td> Trizma de base </td><td> Sigma-Aldrich </td><td> T1503 </td><td></td></tr><tr><td> Dithiothréitol (DTT) </td><td> Pierce </td><td> 20291 </td><td> «Non-pèsent dithiothréitol&quot; </td></tr><tr><td> Iodoacétamide (IAM) </td><td> Sigma-Aldrich </td><td> I1149 </td><td></td></tr><tr><td> Or la trypsine </td><td> Promega </td><td> V5280 </td><td></td></tr><tr><td> Protéines G perles magnétiques, 2.8um </td><td> Invitrogen </td><td> 10004D </td><td></td></tr><tr><td> Tampon phosphate salin (PBS) </td><td> Thermo Fisher Scientific </td><td> L5401 </td><td></td></tr><tr><td> CHAPS </td><td> Thermo Fisher Scientific </td><td> 28300 </td><td></td></tr><tr><td> L&#39;acide formique </td><td> EMD </td><td> 11670 </td><td></td></tr><tr><td> Acétonitrile </td><td> Thermo Fisher Scientific </td><td> A998-1 </td><td></td></tr><tr><td> Acide acétique </td><td> Sigma-Aldrich </td><td> 242853 </td><td></td></tr></tbody></table> Tableau 2: Réactifs <table border="1"><tbody><tr><th> Solution </th><th> Commentaires (optionnel) </th></tr><tr><td> Tris 100 mM pH 8 </td><td></td></tr><tr><td> 9 M d&#39;urée / 30 mM de DTT dans 100 mM de Tris (pH 8) </td><td> doivent être préparés frais chaque fois que </td></tr><tr><td> 500 mM IAM dans 100 mM de Tris (pH 8) </td><td> doivent être préparés frais chaque fois que </td></tr><tr><td> Trypsine 1mg/mL dans 100 mM Tris pH 8 </td><td></td></tr><tr><td> Inhibiteur de la trypsine 1mg/mL dans 100 mM Tris pH 8 </td><td></td></tr><tr><td> Isotopes stables standards mastermix </td><td></td></tr><tr><td> Mastermix anticorps anti-peptide </td><td></td></tr></tbody></table> Tableau 3: Des solutions pour se préparer

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

Il ya un grand besoin pour des analyses quantitatives pour mesurer les protéines. Immunoessais traditionnels sandwichs, largement considéré comme l&#39;étalon-or dans la quantification, sont associées à un coût élevé, de longs délais, et sont pleines de défauts (par exemple, les anticorps hétérophiles, l&#39;interférence des autoanticorps, «effet crochet»). 1 Une technique alternative est l&#39;enrichissement affinités de peptides couplée à la spectrométrie de masse quantitative, communément appelé SISCAPA (Normes des isotopes stables et capture par anticorps anti-peptide). 2 Dans cette technique, l&#39;enrichissement d&#39;affinité de peptides avec une dilution d&#39;isotopes stables et la détection par sélectionnée / multiples réactions de surveillance spectrométrie de masse ( MRS / MRM-MS) fournit une mesure quantitative de peptides en tant que substituts pour leurs protéines respectives. MRS / MRM-MS est bien établie pour la quantification précise de petites molécules 3, 4 et, plus récemment, a été adapté pour mesurer les concentrations de protéines dans le plasma et les lysats cellulaires. 5-7 Pour réaliser la quantification des protéines, ces molécules plus grosses sont digérés peptides composant en utilisant une enzyme comme la trypsine. Un ou plusieurs peptides sélectionnés dont la séquence est unique à la protéine cible de cette espèce (ie &quot;protéotypiques&quot; peptides) sont ensuite enrichies de l&#39;échantillon en utilisant des anticorps anti-peptide et mesurés comme substituts stoechiométrique quantitatifs pour la concentration de protéines dans l&#39;échantillon. Ainsi, couplée à une dilution des isotopes stables (SID) méthodes (c&#39;est à dire un dopés en isotopes stables peptide marqué standard), le SRM / MRM peuvent être utilisés pour mesurer les concentrations de peptides protéotypiques comme substituts pour la quantification des protéines dans des matrices biologiques complexes. Les tests ont plusieurs avantages par rapport aux immunoessais traditionnels. Les réactifs sont relativement moins chers à produire, la spécificité de l&#39;analyte est excellent, les dosages peuvent être fortement multiplexé, l&#39;enrichissement peut être réalisé à partir du plasma soigné (pas de déplétion requis), et la technique se prête à un large éventail de protéines ou des modifications d&#39;intérêt. 8-13 Dans cette vidéo, nous démontrons que le protocole de base adaptée à une plate-forme bille magnétique.

Watch this Scientific Journal Video about Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry at JoVE.com

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Normes des isotopes stables et capture par anticorps anti-peptide (SISCAPA) l&#39;enrichissement affinités couples de peptides avec des isotopes stables de dilution spectrométrie de masse (MRM-MS) pour fournir une mesure quantitative des peptides comme substituts pour leurs protéines respectives. Nous décrivons ici le protocole en utilisant des particules magnétiques dans un format partiellement automatisés.

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 ...

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Biology

22.7K vues.  Fred Hutchinson Cancer Research Center - FHCRC. Normes des isotopes stables et capture par anticorps anti-peptide (SISCAPA) l'enrichissement affinités couples de peptides avec des isotopes stables de dilution spectrométrie de masse (MRM-MS) pour fournir une mesure quantitative des peptides comme substituts pour leurs protéines respectives. Nous décrivons ici le protocole en utilisant des particules magnétiques dans un format partiellement automatisés.

Vidéo: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

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 ...

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Biologie

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 ...

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 ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Dans la quantification in vivo des interactions des protéines G récepteurs couplés utilisant résolution spectrale microscopie à deux photons

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

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 ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identification des complexes protéiques dans<em&gt; Escherichia coli</em&gt; En utilisant séquentielle Purification par affinité peptide en combinaison avec la spectrométrie de masse en tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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 ...

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 ...