Wir danken Olivier Vallon für den Antiserum gegen CF1β. Diese Arbeit wurde von der Max-Planck-Gesellschaft und Zuschüsse von der Deutschen Forschungsgemeinschaft (Schr 617/5-1) und dem Bundesministerium für Bildung und Forschung (Systems Biology Initiative FORSYS, Projekt GoFORSYS) unterstützt.

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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. 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Eine Vernetzung Schritt zur Erfassung transienter Protein-Protein-Interaktionen (QUICK-X), und eine Steuereinheit Niederschläge für ungleiche Niederschläge Wirkungsgrade 6 normalisieren: Wir haben vor kurzem zwei Verbesserungen an der QUICK Ansatz eingeführt. Hier haben wir ein Protokoll, das zwei weitere Verbesserungen der QUICK präsentieren: Zunächst ersetzen wir SILAC 5 für 15 N metabolische Markierung. Die Vorteile sind, dass 15 N metabolische Markierung viel billiger als SILAC ist, wenn 15 N als einfache anorganische Salz versehen ist. Weiterhin mit 15 N metabolische Markierung QUICK können Organismen prototrophen für alle Aminosäuren angewendet werden, wie die meisten Pflanzen, Pilzen und Bakterien. Und schließlich ist Arginin-zu-Prolin Interkonversion inhärent SILAC 5,6 kein Problem für die Quantifizierung von 15 N markierten Peptiden. Beispiele für geeignete Instrumente für die quantitative Auswertung von 15 N Proteomik-Daten sind MSQUANT 12 oder IOMIQS 13. Zweitens stellen wir Affinität Modulation als Mittel zur gezielten Verringerung der Menge an Proteinen Interagieren mit einer gegebenen Zielproteins in einer Probe gegenüber einem anderen. Die Vorteile dieser Vorgehensweise sind, dass sie den Bau von Knockdown-Mutanten, die für einige Modellsysteme sind schwer zu erzeugen oder gar nicht bei wesentlichen Zielproteine ​​erzeugt werden umgeht. Ferner vermeidet sie Fehlinterpretationen durch differentielle Proteinexpression eventuell auftretende als Antwort der Zelle auf Klopfen Niederhalter ein Zielprotein verursacht: wenn andere Proteine ​​herabreguliert sind so gut und mit dem Antiserum für die Immunpräzipitation eingesetzt kreuzreagieren, würden sie interpretiert werden als wahr Interaktionspartner des Zielproteins. Endlich hebt Affinität Modulation die Notwendigkeit der Suche nach einem geeigneten Antikörper-to-Antigen-Verhältnis. Obwohl wir unser Protokoll Chlamydomonas reinhardtii als Modellorganismus, Kann es leicht zu einem anderen Organismus, in Zellkultur gezüchtet werden können und in der Lage ist Ammonium oder Nitrat als Stickstoffquelle zu verwenden angepasst werden. Affinität Modulation von Proteinkomplexen durch ATP / ADP kann direkt an andere Chaperone, deren Wechselwirkung mit Substraten und Kohorte Proteine ​​hängt vom ATP Zustand aufgetragen werden, wie die GroEL/HSP60/Cpn60 oder HSP90 Chaperonsysteme 14,15, oder zu einem anderen System, bei dem Bindungsaffinitäten sind moduliert durch ATP. Affinität Modulation sollte auch für Fälle, in denen Affinitäten zwischen Protein-Wechselwirkungen durch spezifische Medikamente, wie Radicicol oder Geldanamycin im Fall von HSP90-Systeme 15 verändert werden arbeiten. Eine klare Beschränkung unserer Protokoll ist, dass es erfordert affinitätsgereinigten Antikörper gegen ein Zielprotein bekanntlich empfindlich auf eine spezifische Behandlung / Medikament, seine Affinität für Partner Proteine ​​moduliert. Daher ist es nicht Hochdurchsatzverfahren.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> ProteinA Sepharose </td><td> Sigma-Aldrich </td><td> P3391 </td><td></td></tr><tr><td> DMP (Dimethyl pimelimidate) </td><td> Sigma-Aldrich </td><td> D8388 </td><td> Bewahren Sie trocken bei -20 ° C, direkt auflösen, bevor verwenden </td></tr><tr><td> Protease-Inhibitor (komplett, EDTA-free) </td><td> Roche Applied Science </td><td> 11873580001 </td><td></td></tr><tr><td> ATP (Adenosin-5&#39;-triphosphat) </td><td> Carl Roth </td><td> K054 </td><td></td></tr><tr><td> Kreatinphosphat </td><td> Sigma-Aldrich </td><td> 27920 </td><td></td></tr><tr><td> Kreatinphosphokinase </td><td> Sigma-Aldrich </td><td> C7886 </td><td>; </td></tr><tr><td> DSP Dithiobis [Succinimidylpropionat] </td><td> Thermo Sientific </td><td> 22585 </td><td> Bewahren Sie trocken bei 4 ° C </td></tr><tr><td> 15 NH 4 Cl </td><td> Cambridge Isotopen Laboratorien, Andover, MA </td><td> NLM-467 </td><td></td></tr><tr><td> Lys-C (Endoproteinase Lys-C) </td><td> Roche Applied Science </td><td> 11047825001 </td><td></td></tr><tr><td> Trypsin Perlen (Poroszyme immobilisiertem Trypsin) </td><td> Applied Biosystems </td><td> 2-3127-00 </td><td> Gründlich mischen unmittelbar vor Gebrauch </td></tr><tr><td> Empore C 18 47 mm Scheibe </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

Protein-Protein-Wechselwirkungen sind für viele biologische Prozesse in der Zelle von grundlegender Bedeutung. Deshalb spielt deren Charakterisierung eine wichtige Rolle in der gegenwärtigen Forschung und eine Vielzahl von Methoden für ihre Untersuchung steht ein. Protein-Protein Wechselwirkungen sind oft sehr dynamisch und kann auf der subzellulären Lokalisation, posttranslationale Modifizierungen und dem lokalen Proteinumgebung 2 abhängen. Daher sollten sie in ihrer natürlichen Umgebung untersucht werden, für die Co-Immunopräzipitation Ansätze die Methode der Wahl 3 sind. Co-gefällte Interaktionspartner werden entweder durch Immunoblotting in einem zielgerichteten Ansatz oder durch Massenspektrometrie (LC-MS/MS) in einer ungezielte Weise identifiziert. Die letztgenannte Strategie wird oft negativ von einer großen Zahl von falsch positiven Entdeckungen, vor allem aus der hohen Empfindlichkeit der modernen Massenspektrometern, die selbstbewusst erkennt Spuren von unspezifisch Ausfällen von Proteinen abgeleitet betroffen. A recent Ansatz zur Überwindung dieses Problems liegt der Gedanke zugrunde, dass verringerte Mengen an spezifischen Bindungspartner wird Copräzipitat mit einer gegebenen Zielprotein deren zelluläre Konzentration wird durch RNAi reduziert, während die Mengen an unspezifisch Ausfällen von Proteinen sein sollte unberührt. Dieser Ansatz, genannt QUICK zur quantitativen Immunpräzipitation mit Knockdown 4 Kombinierte beschäftigt Stable Isotope Labeling von Aminosäuren in Zellkultur (SILAC) 5 und MS, um die Mengen von Proteinen aus Wildtyp-und knock-down Stämme immunpräzipitiert quantifizieren. Proteinen in einem Verhältnis von 1:1 zu finden ist als Verunreinigungen, die in Ausscheidungen vom Wildtyp als spezifische Bindungspartner des Zielproteins angereichert betrachtet werden. Obwohl innovative trägt QUICK einige Einschränkungen: Erstens ist SILAC kostenintensiv und beschränkt auf Organismen, die im Idealfall auxotroph für Arginin und / oder Lysin. Außerdem, wenn schwere Arginin eingespeist wird, Arginin-zu-Prolin Umwandlung führt additional Masse verschiebt für jeden Prolin in einem Peptid und leicht verdünnt schwere mit Licht Arginin, die Quantifizierung mühsamer und weniger genau 5,6 macht. Zweitens erfordert QUICK dass Antikörper titriert sind, dass sie nicht in die Sättigung mit Zielprotein in Extrakten aus knock-down-Mutanten. Hier stellen wir ein modifiziertes Quick-Protokoll, das die obengenannten Einschränkungen von SCHNELL durch Ersetzen SILAC für 15 N metabolische Markierung und durch Ersetzen RNAi-vermittelte knock-down für Affinität Modulation von Protein-Protein-Wechselwirkungen überwindet. Wir demonstrieren die Anwendbarkeit dieses Protokolls unter Verwendung der einzelligen Grünalge Chlamydomonas reinhardtii als Modellorganismus und der Chloroplasten HSP70B Chaperon als Zielprotein 7 (Abbildung 1). HSP70s sind dafür bekannt, mit speziellen Co-Chaperone und Substrate nur in der ADP Zustand 8 interagieren. Wir nutzen diese Eigenschaft als ein Mittel, um den spezifischen überprüfenZusammenspiel von HSP70B mit seinen nucleotide exchange factor 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

Wir präsentieren eine Variation der QUICK (quantitativen Immunpräzipitation mit Knockdown kombiniert) Ansatz, der zuvor wurde eingeführt, um zwischen wahr und falsch Protein-Protein-Wechselwirkungen zu unterscheiden. Unser Ansatz basiert auf<sup&gt; 15N</sup&gt; Metabolische Markierung, die Modulation der Affinitäten von Protein-Protein-Wechselwirkungen durch die Anwesenheit / Abwesenheit von ATP, Immunpräzipitation und quantitative Massenspektrometrie.

Ein Protokoll für die Identifizierung von Protein-Protein-Wechselwirkungen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung, Immunpräzipitation, Quantitative Massenspektrometrie und Affinity 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 Aufrufe.  Max Planck Institute of Molecular Plant Physiology. Wir präsentieren eine Variation der QUICK (quantitativen Immunpräzipitation mit Knockdown kombiniert) Ansatz, der zuvor wurde eingeführt, um zwischen wahr und falsch Protein-Protein-Wechselwirkungen zu unterscheiden. Unser Ansatz basiert auf 15N Metabolische Markierung, die Modulation der Affinitäten von Protein-Protein-Wechselwirkungen durch die Anwesenheit / Abwesenheit von ATP, Immunpräzipitation und quantitative Massenspektrometrie.

Serie: 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).

MALDI-Probenvorbereitung: Die Ultra Thin-Layer-Methode

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Forschung

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.

Eine quantitative Bewertung der Hefe Lipidome mit Elektrospray-Ionisation-Massenspektrometrie

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.

Identifizierung von Protein-Komplexen in<em&gt; Escherichia coli</em&gt; Mit Sequential Peptide Affinity Purification in Kombination mit Tandem-Massenspektrometrie

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.

Ein neuer Ansatz für die vergleichende Analyse von Multiproteinkomplexen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung und quantitative Massenspektrometrie

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.

Untersuchung von Protein-Protein-Interaktionen in lebenden Zellen Mit Biolumineszenz-Resonanz-Energie-Transfer

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.

Chromatinimmunpräzipitation Assay zur Identifizierung von Arabidopsis-Protein-DNA-Wechselwirkungen<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.

Identifizierung von MyoD Interaktom Gekoppelt Mit Tandem Affinity Purification zu Mass Spectrometry

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.

Untersuchung von Protein-Protein-Interaktionen in der Autophagie-Forschung

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.

Quantifizierung der standortspezifischen Protein Lysin Acetylierung und Succinylation Stöchiometrie mittels Massenspektrometrie Daten-unabhängige Übernahme

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.

SA--Galactosidase-basierter Screening-Test zur Identifizierung von Senotherapeutischen Arzneimitteln

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 ...