Nous tenons à remercier Olivier Vallon pour l&#39;antisérum contre CF1β. Ce travail a été soutenu par la Société Max Planck et des subventions de la Deutsche Forschungsgemeinschaft (Schr 617/5-1) et le Bundesministerium für Bildung und Forschung (biologie des systèmes Forsys Initiative, GoFORSYS projet).

<ol>
	<li>Perrakis, A., Musacchio, A., Cusack, S., Petosa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+a+macromolecular+complex:+the+toolkit+of+methods.">Investigating a macromolecular complex: the toolkit of methods.</a> J. Struct. Biol. 175, 106-112 (2011).</li><li>Gavin, A. C., Maeda, K., Kuhner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+charting+protein-protein+interaction:+mass+spectrometry-based+approaches.">Recent advances in charting protein-protein interaction: mass spectrometry-based approaches.</a> Curr. Opin. Biotechnol. 22, 42-49 (2011).</li><li>Markham, K., Bai, Y., Schmitt-Ulms, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-immunoprecipitations+revisited:+an+update+on+experimental+concepts+and+their+implementation+for+sensitive+interactome+investigations+of+endogenous+proteins.">Co-immunoprecipitations revisited: an update on experimental concepts and their implementation for sensitive interactome investigations of endogenous proteins.</a> Anal. Bioanal. Chem. 389, 461-473 (2007).</li><li>Selbach, M., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+interaction+screening+by+quantitative+immunoprecipitation+combined+with+knockdown+(QUICK.">Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK.</a> Nat Methods. 3, 981-983 (2006).</li><li>Ong, S. E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+recipe+for+stable+isotope+labeling+by+amino+acids+in+cell+culture+(SILAC).">A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).</a> Nat Protoc. 1, 2650-2660 (2006).</li><li>Heide, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+quantitative+immunoprecipitation+combined+with+knockdown+and+cross-linking+to+Chlamydomonas+reveals+the+presence+of+vesicle-inducing+protein+in+plastids+1+in+a+common+complex+with+chloroplast+HSP90C.">Application of quantitative immunoprecipitation combined with knockdown and cross-linking to Chlamydomonas reveals the presence of vesicle-inducing protein in plastids 1 in a common complex with chloroplast HSP90C.</a> Proteomics. 9, 3079-3089 (2009).</li><li>Nordhues, A., Miller, S. M., Muhlhaus, T., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+roles+of+molecular+chaperones+in+Chlamydomonas+and+Volvox.">New insights into the roles of molecular chaperones in Chlamydomonas and Volvox.</a> International review of cell and molecular biology. 285, 75-113 (2010).</li><li>Mayer, M. P., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperones:+cellular+functions+and+molecular+mechanism.">Hsp70 chaperones: cellular functions and molecular mechanism.</a> Cell Mol. Life Sci. 62, 670-684 (2005).</li><li>Schroda, M., Vallon, O., Whitelegge, J. P., Beck, C. F., Wollman, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chloroplastic+GrpE+homolog+of+Chlamydomonas:+two+isoforms+generated+by+differential+splicing.">The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing.</a> The Plant Cell. 13, 2823-2839 (2001).</li><li>Willmund, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEAT+SHOCK+PROTEIN+90C+is+a+bona+fide+Hsp90+that+interacts+with+plastidic+HSP70B+in+Chlamydomonas+reinhardtii.">HEAT SHOCK PROTEIN 90C is a bona fide Hsp90 that interacts with plastidic HSP70B in Chlamydomonas reinhardtii.</a> Plant Physiol. 138, 2310-2322 (2005).</li><li>Harris, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use. , (2008).</li><li>Mortensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSQuant,+an+open+source+platform+for+mass+spectrometry-based+quantitative+proteomics.">MSQuant, an open source platform for mass spectrometry-based quantitative proteomics.</a> J. Proteome Res. 9, 393-403 (2010).</li><li>M?hlhaus, T., Weiss, J., Hemme, D., Sommer, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Shotgun+Proteomics+Using+a+Uniform+15N-Labeled+Standard+to+Monitor+Proteome+Dynamics+in+Time+Course+Experiments+Reveals+New+Insights+into+the+Heat+Stress+Response+of+Chlamydomonas+reinhardtii.">Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii.</a> Mol. Cell Proteomics. 10, (2011).</li><li>Bukau, B., Horwich, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp70+and+Hsp60+chaperone+machines.">The Hsp70 and Hsp60 chaperone machines.</a> Cell. 92, 351-366 (1998).</li><li>Wandinger, S. K., Richter, K., Buchner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp90+chaperone+machinery.">The Hsp90 chaperone machinery.</a> J. Biol. Chem. 283, 18473-18477 (2008).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Nous avons récemment introduit deux améliorations à l&#39;approche RAPIDE: une étape de réticulation pour la capture transitoires interactions protéine-protéine (QUICK-X), et une précipitation de contrôle pour normaliser les efficacités des précipitations inégales 6. Nous présentons ici un protocole contenant deux autres améliorations de QUICK: tout d&#39;abord, nous remplaçons SILAC 5 pour 15 N marquage métabolique. Les avantages sont que 15 N marquage métabolique est beaucoup moins cher que SILAC, si 15 N est fourni sous forme de sel inorganique simple. En outre, avec 15 N métabolique étiquetage RAPIDE peut être appliqué à des organismes prototrophes pour tous les acides aminés, comme la plupart des plantes, les champignons et les bactéries. Et enfin, l&#39;arginine-à-proline interconversion inhérent à 5,6 SILAC ne pose pas de problème pour la quantification de 15 N peptides marqués. Exemples d&#39;outils appropriés pour l&#39;évaluation quantitative de la protéomique N 15 données sont 12 MSQUANT ou jeOMIQS 13. Deuxièmement, on introduit une modulation d&#39;affinité en tant que moyen pour réduire spécifiquement la quantité de protéines qui interagissent avec une protéine cible dans un échantillon donné par rapport à l&#39;autre. Les avantages de cette approche est qu&#39;elle contourne la construction de mutants knock-down, qui, pour certains systèmes modèles sont difficiles à produire ou ne peut pas être généré du tout dans le cas de protéines cibles essentielles. En outre, elle permet d&#39;éviter des interprétations erronées causées par l&#39;expression différentielle des protéines potentiellement se produisant en réponse à la cellule de frappe vers le bas d&#39;une protéine cible: si d&#39;autres protéines sont régulés à la baisse et ainsi une réaction croisée avec l&#39;antisérum utilisé pour l&#39;immunoprécipitation, ils sont interprétés comme de véritables partenaires d&#39;interaction de la protéine cible. Enfin, l&#39;affinité de modulation supprime la nécessité de trouver un bon anticorps-antigène au rapport. Bien que nous appliquons notre protocole de Chlamydomonas reinhardtii comme organisme modèle, Il peut facilement être adapté à tout autre organisme qui peut être cultivée en culture cellulaire et est capable d&#39;utiliser d&#39;ammonium ou de nitrate comme source d&#39;azote. Modulation de l&#39;affinité des complexes protéiques par l&#39;ATP / ADP peut être directement appliquée à d&#39;autres chaperons dont l&#39;interaction avec des substrats et des protéines de cohorte dépend de l&#39;état ATP, comme les GroEL/HSP60/Cpn60 ou HSP90 systèmes chaperon 14,15, ou à tout autre système où affinités de liaison sont modulés par l&#39;ATP. Affinity modulation devrait aussi fonctionner pour les cas où les affinités entre les interactions entre protéines sont altérés par des médicaments spécifiques, comme la geldanamycine radicicol ou dans le cas de systèmes de HSP90 15. Une limitation claire de notre protocole est qu&#39;il nécessite purifié par affinité des anticorps dirigés contre une protéine cible connue pour être sensible à un traitement spécifique / médicament qui module son affinité pour les protéines partenaires. Par conséquent, il n&#39;existe pas de méthode à haut débit.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Proteína Sepharose </td><td> Sigma-Aldrich </td><td> P3391 </td><td></td></tr><tr><td> DMP (diméthyl pimelimidate) </td><td> Sigma-Aldrich </td><td> D8388 </td><td> Stockez desséchée à -20 ° C, se dissolvent directement avant utilisation </td></tr><tr><td> Inhibiteur de la protéase (complet, sans EDTA) </td><td> Roche Applied Science </td><td> 11873580001 </td><td></td></tr><tr><td> ATP (adénosine-5&#39;-triphosphat) </td><td> Carl Roth </td><td> K054 </td><td></td></tr><tr><td> La créatine phosphate </td><td> Sigma-Aldrich </td><td> 27920 </td><td></td></tr><tr><td> Créatine phosphokinase </td><td> Sigma-Aldrich </td><td> C7886 </td><td>; </td></tr><tr><td> DSP dithiobis [succinimidyl propionate] </td><td> Sientific Thermo </td><td> 22585 </td><td> Stockez desséchée à 4 ° C </td></tr><tr><td> 15 NH 4 Cl </td><td> Cambridge Isotope Laboratories, Andover, MA </td><td> NLM-467 </td><td></td></tr><tr><td> Lys-C (endoprotéinase Lys-C) </td><td> Roche Applied Science </td><td> 11047825001 </td><td></td></tr><tr><td> Perles trypsine (trypsine immobilisée Poroszyme) </td><td> Applied Biosystems </td><td> 2-3127-00 </td><td> Mélanger vigoureusement juste avant d&#39;utiliser </td></tr><tr><td> Disque Empore mm 18 C 47 </td><td> Varian </td><td> 12145004 </td><td></td></tr></tbody></table>

a protocol for the identification of protein-protein interactions based on 15n metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation

Interactions protéine-protéine sont fondamentales pour de nombreux processus biologiques dans la cellule. Par conséquent, leur caractérisation joue un rôle important dans la recherche actuelle et une multitude de méthodes pour leur enquête est disponible 1. Interactions protéine-protéine sont souvent hautement dynamique et peut dépendre de la localisation subcellulaire, modifications post-traductionnelles des protéines et de l&#39;environnement local 2. Par conséquent, ils devraient être étudiés dans leur environnement naturel, pour lesquels les approches de co-immunoprécipitation sont la méthode de choix 3. Partenaires d&#39;interaction co-précipités sont identifiés soit par immunoblot dans une approche ciblée, ou par spectrométrie de masse (LC-MS/MS) d&#39;une manière non ciblée. Cette dernière stratégie est souvent affectée par un grand nombre de fausses découvertes positives, provient principalement de la forte sensibilité des spectromètres de masse modernes confiance détecter des traces de protéines précipitant non spécifique. Un recent approche pour résoudre ce problème est basée sur l&#39;idée que des quantités réduites de partenaires d&#39;interaction spécifiques sera co-précipité avec une protéine cible donnée dont la concentration cellulaire est réduite par l&#39;ARNi, tandis que les quantités de protéines non spécifique de précipitation devraient pas être affectés. Cette approche, appelée RAPIDE pour immunoprécipitation quantitative Combiné avec Knockdown 4, emploie étiquetage des isotopes stables d&#39;acides aminés dans la culture cellulaire (SILAC) 5 et MS pour quantifier les quantités de protéines immunoprécipitées à partir de souches de type sauvage et knock-down. Protéines présentes dans un rapport de 1:1 peut être considéré comme contaminants, enrichie en ces précipités du type sauvage comme partenaires d&#39;interaction spécifique de la protéine cible. Bien que novateur, QUICK porte certaines limites: d&#39;une part, SILAC est très coûteux et limité pour les organismes qui, idéalement, sont auxotrophes pour l&#39;arginine et / ou la lysine. Des résultats d&#39;interconversion De plus, lorsque l&#39;arginine lourde est alimenté, l&#39;arginine-à-proline dans additional de masse se déplace pour chaque proline dans un peptide et dilue un peu lourd avec de l&#39;arginine lumière, ce qui en fait une quantification plus fastidieux et moins précis 5,6. D&#39;autre part, RAPIDE nécessite que des anticorps sont titrés de manière à ne pas se saturer avec la protéine cible dans des extraits de mutants knock-down. Ici, nous introduisons un protocole modifié QUICK qui surmonte les limitations mentionnées ci-dessus en remplaçant de QUICK SILAC pour l&#39;étiquetage 15 N métabolique et en remplaçant l&#39;ARNi médiée par le knock-down pour la modulation de l&#39;affinité interactions protéine-protéine. Nous démontrons l&#39;applicabilité de ce protocole en utilisant les algue verte unicellulaire Chlamydomonas reinhardtii comme organisme modèle et le chaperon hsp70B chloroplaste de 7 protéine cible (figure 1). HSP70s sont connus pour interagir avec certains co-chaperons et des substrats que dans l&#39;état ADP 8. Nous exploitons cette propriété comme un moyen de vérifier le spécifiqueinteraction de hsp70B avec son facteur d&#39;échange des nucléotides CGE1 9.

Watch this Scientific Journal Video about A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Aff

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Nous présentons une variante de l&#39;RAPIDE (immunoprécipitation quantitative Combiné avec Knockdown) approche qui a déjà été introduite pour distinguer entre les vraies et les fausses interactions protéine-protéine. Notre approche est basée sur<sup&gt; 15N</sup&gt; Marquage métabolique, la modulation des affinités interactions protéine-protéine par la présence / absence d&#39;ATP, immunoprécipitation et spectrométrie de masse quantitative.

Un protocole pour l&#39;identification des interactions protéine-protéine Basé sur<sup&gt; 15</sup&gt; N marquage métabolique, immunoprécipitation, la spectrométrie de masse quantitative et d&#39;affinité de modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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Research

JoVE Journal

Biology

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

20.4K vues.  Max Planck Institute of Molecular Plant Physiology. Nous présentons une variante de l'RAPIDE (immunoprécipitation quantitative Combiné avec Knockdown) approche qui a déjà été introduite pour distinguer entre les vraies et les fausses interactions protéine-protéine. Notre approche est basée sur 15N Marquage métabolique, la modulation des affinités interactions protéine-protéine par la présence / absence d'ATP, immunoprécipitation et spectrométrie de masse quantitative.

Vidéo: A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

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

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 ...

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 ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Une nouvelle approche pour l&#39;analyse comparative des complexes multiprotéiques Basé sur<sup&gt; 15</sup&gt; N Marquage métabolique et spectrométrie de masse quantitative

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Étudier les interactions protéine-protéine dans les cellules vivantes en utilisant le transfert d&#39;énergie de résonance de bioluminescence

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprécipitation de la chromatine test pour l&#39;identification de Arabidopsis interactions protéine-ADN<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Identification de MyoD Interactome Utilisation Tandem Affinity Purification couplée à la spectrométrie de masse

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

Étude des Interactions protéine-protéine dans la recherche de l’autophagie

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Quantification des protéines spécifiques au site Lysine acétylation et stoechiométrie Succinylation spectrométrie de masse indépendante des données Acquisition

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Essai de dépistage basé sur sA-MD-Galactosidase pour l'identification des médicaments sérothérapeutiques

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...