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

1. Lysatherstellung <ol><li> Wachsen P. aeruginosa-Zellen in LB an die gewünschte OD. HINWEIS: Für Hinweise: Verwenden ≈ 100 ml Kultur / Probe für stationäre Phase Kulturen und ≈ 500 ml cultur / Probe für log-Phase Kulturen (OD 600nm = 0,5). </li><li> Pellet durch Zentrifugation für 20 min bei 5.000 x g. </li><li> Resuspendieren 0,5-1 g Pellet in 1 ml Lyse-Puffer (6,7 mM MES, 6,7 mM HEPES, 200 mM NaCl, 6,7 mM KAc, 1 mM DDT, pH 7,5) und füge einen Proteaseinhibitor (vollständige Mini, EDTA-frei) sowie als DNaseI. </li><li> Lyse der Zellen durch 3 Passagen durch einen Französisch Druckzelle bei 20.000 psi (siehe Materialliste). </li><li> Ultrazentrifuge das Zelllysat bei 100.000 x g für 1 h bei 4 ° C. </li><li> Speichern Sie den Überstand (gehen Sie zu Schritt 2). </li><li> Durch Auf- und Abpipettieren Waschen des Pellets mit 1 ml 1x Lysepuffer. </li><li> Ultrazentrifuge bei 100,000 x g für 1 h bei 4 ° C. </li><li> Flash-einfrieren des Pellets inflüssigem Stickstoff und bei -20 ° C bis zur Erfassung von Membranproteinen (siehe Schritt 3). </li></ol> 2. Entfernung von Freier c-di-GMP und andere Nukleotide (lösliche Fraktion Only) <ol><li> Mit 10 ml kaltem Lysepuffer Waschen eine PD10-Entsalzungssäule (siehe Materialliste). </li><li> Gießen Sie den Überstand (≈ 1 ml) auf die PD10, um Nukleotide zu entfernen. </li><li> Eluiert mit 4 ml kaltem Lysepuffer (500 ul Stufen). </li><li> Wählen Sie die am stärksten konzentrierten Fraktionen wie von Bradford bestimmt und sie gemeinsam. </li></ol> 3. Pellet Resuspension und Solubilisierung (Membranfraktion Only) <ol><li> Das Pellet in 500 bis 1000 ul 1x Sammlungspuffer (ohne Reinigungsmittel) (siehe Tabelle 1). HINWEIS: Das Pellet ist schwierig, wieder zu suspendieren. Es ist mit der ersten Pipette auf und ab mit einer Pipette wird empfohlen, etwa das Pellet, dann, eine Spritze 27 G zu verwenden, um auch die Lösung zu homogenisieren. </li><li> 1% (w / v) n-Dodecyl-β-D-maltopyranosid (DDM). </li><li> Inkubieren bei 4 ° C für mindestens 2 h (oder O / N) auf einem rotierenden Rad. </li><li> Ultrazentrifuge bei 100,000 x g für 1 h bei 4 ° C. </li><li> Ernten Sie den Überstand. </li></ol> 4. Proteinkonzentrationsmessung <ol><li> Messung der Proteinkonzentration durch Bradford-Assays (BCA-Assay für Membranfraktion). </li><li> Stellen Sie die Gesamtproteinkonzentration auf 10 mg / ml. </li></ol> 5. Erfassen <ol><li> Mischungs 300 ug Protein und mit 1 mM GDP, GTP, ATP, CTP und 20 ul 5x Aufnahmepuffer (100 mM HEPES, 250 mM KAc, 50 mM MgAc, 5% Glycerin, pH 7,5) (Tabelle 1). Stellen Sie das Reaktionsvolumen auf 100 ul mit H 2 O. HINWEIS: das Gesamtvolumen umfasst das Volumen des CDG-CC und c-di-GMP bei der Wettbewerbskontrolle (siehe Schritt 5.3 und Tabelle 2). HINWEIS: Alle Experimente wurden in 200 &amp; geführt# 181; l 12-Rohr-PCR-Streifen (Thermo Scientific). HINWEIS: für die Membranfraktion, stellen Sie sicher, immer die DDM-Konzentration zu halten über der kritischen Mizellenkonzentration (0,01% w / v), bis das Protein Solubilisierungsschritt in 8 M Harnstoff (Schritt 7.1). </li><li> Inkubieren bei 4 ° C für 30 min auf einem sich drehenden Rad. </li><li> In 10 uM CDG-CC (Endkonzentration). HINWEIS: eine Steuer ohne CDG-CC (als &quot;Perle Steuerung&quot; bezeichnet), und eine Steuer mit 1 mM c-di-GMP (&quot;Wettbewerbskontrolle&quot;) ergänzt (siehe Tabelle 2). Hinweis: Die CDG-CC-Konzentration von 1 bis 10 &amp; mgr; M eingestellt werden. </li><li> Inkubieren bei 4 ° C für mindestens 2 h (O / N für die Membranfraktion) auf einem rotierenden Rad, in der Dunkelheit. </li><li> Nach einer kurzen Spin, vernetzen durch Aktivierung des reaktiven Rest des CDG-CC mit UV-Licht für 4 Minuten mit einem CaproBox (siehe Materialliste, λ = 310 nm, Bestrahlungsstärke ≥10 mW / cm², Entfernung von der Quelle = 2 cm). HINWEIS: Entfernen Sie den Deckel der Streifen vor der Vernetzung. </li><li> Man gebe 25 &amp; mgr; l 5x-Waschpuffer (5 M NaCl, 250 mM Tris, pH 7,5) und 50 ul gut resuspendierten Streptavidin-Magnetkügelchen, sanft zu homogenisieren. </li><li> Inkubieren bei 4 ° C für 1 h auf einem rotierenden Rad. </li></ol> 6. Waschschritte HINWEIS: (Magnet: siehe Materialliste). Beginnen Sie mit einer Erfassung der magnetischen Kügelchen in der PCR-Streifen Deckel, mit dem Magneten. Dann ersetzen Sie die PCR Streifen durch eine neue mit dem nächsten Waschlösung. Entfernen Sie den Magneten und resuspendieren die Perlen, und Inkubation 2 min. Spin down, und ersetzen Sie den Deckel durch eine frische Deckel. <ol><li> Waschschritte (lösliche Fraktion nur) <ol><li> 6-mal in 200 ul 1x Waschpuffer waschen. </li><li> Einmal in 200 ul HPLC-Qualität H 2 O Waschen </li><li> 6-mal in 200 ul 80% Acetonitril waschen. </li><li> Waschen Sie 2-mal in 200 ul HPLC-Qualität H 2 O </li></ol></li><li> Waschschritte (membrane Bruch nur) <ol><li> 5 mal in 200 ul 1x Waschpuffer + 0.1% DDM waschen. </li><li> In 200 ul 1x Waschpuffer + 0,05% DDM waschen 2 mal. </li><li> Einmal in 200 ul 1x Waschpuffer + 0,025% DDM waschen. </li><li> Einmal in 200 ul 1x Waschpuffer + 0,0125% DDM waschen. </li><li> 3 Mal Waschen in 200 ul 100 mM ABC (Ammoniumbicarbonat, NH 4 CO 3) + 2 M Harnstoff. </li></ol></li></ol> 7. MS Probenvorbereitung <ol><li> Resuspendieren der Beads (direkt im Deckel) in 20 ul 100 mM ABC (100 mM ABC + 8 M Harnstoff für die Membranfraktion) und Überführung in 1,5-ml-Röhrchen. </li><li> Nur Membranfraktion: Inkubation bei 60 ° C für 5 min, bei 500 Upm Schütteln. </li><li> In 0,5 ul 200 mM TCEP (Tris (2-Carboxyethyl) phosphin) und Inkubation bei 60 ° C für 1 h, bei 500 Upm schüttelnd. Abkühlen auf 25 ° C. </li><li> In 0,5 ul frisch hergestelltes 400 mM Iodacetamid und Inkubation bei 25 ° C für 30 min, Schütteln einT 500 Umdrehungen pro Minute und in der Dunkelheit. </li><li> Mit 0,5 ul 0,5 M N-Acetyl-Cystein und Inkubieren bei 25 ° C für 10 min, bei 500 Upm Schütteln. </li><li> Nur Membranfraktion: fügen 1 ul Lys-C und bei 37 ° C, O / N. </li><li> In 2 ug Trypsin und Inkubation O / N bei 37 ° C, bei 500 Upm (wrap in Parafilm Austrocknen zu verhindern) Schütteln. HINWEIS: Die Proben können bei -20 ° C zu diesem Zeitpunkt gespeichert. HINWEIS: Membranfraktion nur: mit 100 mM ABC, um die Harnstoffkonzentration auf &lt;2 M vor der Zugabe von Trypsin anpassen. </li><li> Kurz Spin-Down der Rohre und sammeln Kügelchen mit dem Magneten. </li><li> Den Überstand in ein neues 1,5-ml-Röhrchen (wiederholen Sie diesen Schritt, wenn Perlen gelassen werden). </li><li> Mit 5 &amp; mgr; l 5% TFA (Trifluoressigsäure) + 1 &amp; mgr; l 2 M HCl (15 &amp; mgr; l 5% TFA + 5 &amp; mgr; l 2 M HCl für die Membranfraktion). </li><li> Bedingung C18 Microspin-Säulen (The Nest Group, MA, USA) mit 150 &amp; mgr; l Acetonitril (spin 20 sec bei 2400 rpm). </li><li> Equilibrate der C18-Säulen 2 mal mit 150 ul 0,1% TFA (Spin 20 Sekunden, 2400 rpm). </li><li> Laden Sie die Probe und Spin 2 min, 2000 Upm. </li><li> Neuladen des Durchfluss auf die Säule und wiederholen den Spinnschritt (2 min, 2.000 rpm). </li><li> 3x waschen mit 150 ul 0,1% TFA, 5% Acetonitril (20 sec, 2.400 rpm). </li><li> Nehmen Sie eine neue Röhre und eluieren zweimal mit 150 ul 0,1% TFA, 50% Acetonitril (2 min, 2.000 rpm). </li><li> Man trocknet die Peptide in einer Speed-vac. </li><li> Resuspendieren in 40 &amp; mgr; l 98% H 2 O, 2% Acetonitril, 0,15% Ameisensäure. </li><li> Ultraschallbehandlung 20 s (Impulszyklus 0,5, Amplitude 100%, siehe Materialliste) und Spin-down 5 s, 12.000 min (Tischzentrifuge). Vortex 10 Sekunden, und Spin-down 5 s, 12.000 min. Übertragen Sie in einem HPLC-Ampulle für LC-MS / MS-Analyse. </li><li> Bei -20 ° C. HINWEIS: Die Proben können bei -20 ° C zu diesem Zeitpunkt gespeichert. </li></ol> 8. LC-MS / MS-Analyse <ol><li> Führen Sie einen Nano-LC (nano-LC-Systeme) equipped mit einer RP-HPLC-Säule (75 um x 37 cm) mit C18 Harz gepackt (Magic C18 AQ 3 &amp; mgr; m) unter Verwendung eines linearen Gradienten von 95% Lösungsmittel A (0,15% Ameisensäure, 2% Acetonitril) und 5% Lösungsmittel B ( 98% Acetonitril, 0,15% Ameisensäure) bis 35% Lösungsmittel B über 60 min bei einer Flussrate von 0,2 ul / min. </li><li> Analyse der Peptide mittels LC-MS / MS (Zweidruck LTQ-Orbitrap Velos Massenspektrometer, mit einem Elektrospray-Ionenquelle verbunden ist). HINWEIS: Die Datenaufnahme-Modus wurde eingestellt, um eine hochauflösende MS-Scan in der FT Teil des Massenspektrometers mit einer Auflösung von 60.000 FWHM gefolgt von MS / MS-Scans in der linearen Ionenfalle der 20 intensivsten Ionen zu erhalten. Um die Effizienz der MS / MS-Versuche zu erhöhen, wurde der Ladezustand Screening Modus aktiviert werden, damit nicht zugeordnete auszuschließen und einzeln berechnet Ionen. Collusion induzierte Dissoziation wurde ausgelöst, als der Vorläufer über 100 Ionen zählt. Die dynamische Ausschluss Dauer wurde auf 30 Sekunden eingestellt. Der Ionenakkumulationszeit auf 300 ms (MS) und 50 eingestelltms (MS / MS). </li></ol> 9. Datenbanksuche <ol><li> Laden Sie die P. aeruginosa NCBI-Datenbank über die NCBI-Homepage ( <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ). </li><li> Konvertieren Sie die MS Rohspektren in Maskottchen Generika-Dateien (mp) mit dem MassMatrix Konvertierungstool ( <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a> ). </li><li> Suche auf der mp-Datei mit MASCOT Version 2.3 gegen die P. aeruginosa NCBI-Datenbank, die vorwärts und rückwärts-Köder-Protein-Einträge. </li><li> Führen Sie ein in silico Trypsinverdau nach Lysin und Arginin (es sei denn, durch Prolin gefolgt) tolerieren zwei verpasst Spaltungen in voll tryptischen Peptide. </li><li> Stellen Sie die Datenbanksuchparameter, um oxidierte Methionine (15,99491 Da) als variable Änderungen und Carboxyamidomethylierung (57,021464 Da) von Cysteinresten als feste Modifikation kann. Für MASCOT sucht unsing hochauflösende Scans, stellen Sie den Vorläufer Massentoleranz bis 15 ppm und stellen Sie die Fragmentmassentoleranz von 0,6 Da. Schließlich, stellen Sie den Protein FDR auf 1%. </li><li> Importieren Sie die Maskottchen-Durchsuchungen von P. aeruginosa CCMS Experimente in Scaffold (Proteomesoftware, Version 3), stellen Sie die Parameter, um ein Protein FDR knapp 1% zu erhalten, und extrahieren Sie die gesamte spektrale zählt. </li><li> Für in diesem Papier die repräsentativen Ergebnisse präsentiert, haben wir eine gepaarte T-Test, den Versuch mit der Wettbewerbskontrolle zu vergleichen und nur dann als Treffer mit einem p-Wert von weniger als 0,1 und einer spektralen Zahlverhältnis über 2 (experimentieren spektrale counts / Wettbewerbskontrolle Spektralbereich Aktivität). </li><li> Die Daten können in jedem Tabellenkalkulations-Software zur weiteren Analyse exportiert werden. </li></ol> 10. Markierungsfreie Quantifizierung <ol><li> Importieren Sie die Raw-Dateien in Progenesis LC-MS-Software (Nichtlineare Dynamik, Version 4.0). </li><li> Führen LC-MS Ausrichtung und Feature-Erkennung in Verzug settings. </li><li> Exportieren Sie die Daten in mgf Format von Progenesis LC-MS. </li><li> Durchsuchen Sie die MS / MS-Spektren mit Hilfe des MASCOT gegen die NCBI P. aeruginosa Datenbank, die vorwärts und rückwärts-Köder-Protein-Einträge. </li><li> Importieren Sie die Datenbanksuchergebnissen in Progenesis LC-MS und Karte der Peptide Identifikationen zu MS1 Funktionen. </li><li> Für die Datenauswertung (Berechnung der Signifikanzniveaus, klappbar Änderungsverhältnisse) die ProteinSQAnalysis Skript SafeQuant verwendet (Schwelle: q-Wert unter 0,1, spektrale Zahlverhältnis über 2). </li></ol>

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The authors have nothing to disclose.

Besondere Sorgfalt sollte bei verschiedenen Stufen des Protokolls gefaßt. Die Proteinkonzentration ist ein kritischer Parameter bei einer Konzentration von 10 mg / ml, schwierig zu erreichen, wenn die Zellen unter bestimmten Wachstumsbedingungen (zB Biofilmen oder kleine Kolonie Varianten) gezüchtet. Somit sollte das Pellet resuspendiert in einem niedrigen Volumen Lysepuffer geführt werden. Proteinkonzentrationen können bis zu 8 mg / ml reduziert werden. Im Vergleich zu der durch Nespers et al. Ver...

c-di-GMP ist ein wichtiger sekundärer Botenstoff von den meisten Bakterien verwendet, um verschiedene Aspekte des Wachstums und des Verhaltens zu steuern. Zum Beispiel regelt c-di-GMP Zellzyklusprogression, Motilität und die Expression von Exopolysacchariden und Oberflächen Adhäsine 2-4. Durch die Koordination solcher Prozesse c-di-GMP fördert Biofilmbildung, ein Verfahren, das mit einer chronischen Infektion einer Reihe von pathogenen Bakterien 5 zugeordnet ist. c-di-GMP wird durch Enzyme genannt diguanylate Cyclasen (DGCs), die eine katalytische Domäne GGDEF 4 Hafen synthetisiert. Einige DGCs besitzen eine hemmende Website, die nach unten reguliert die Cyclase-Aktivität bei der c-di-GMP verbindlich. Der Abbau von c-di-GMP wird durch zwei unterschiedliche Klassen von Phosphodiesterasen (PDEs) beherbergen entweder eine katalytische EAL oder HD-GYP Domain 6,7 katalysiert. Die Mehrzahl der bekannten Effektorproteine, die direkt binden c-di-GMP gehören zu einer von drei Klassen von Proteinen: katalytischeVerbündeter inaktiv GGDEF oder EAL Domains und PilZ Domains, kleine molekulare Schalter, die Konformationsänderungen bei der c-di-GMP verbindlich 8 unterzogen werden. DGCs, PDEs und PilZ Proteine ​​sind gut gekennzeichnet und ihre Domains können in silico relativ sicher vorhergesagt werden. Ein besonderes Interesse gilt nun auf der Identifizierung von neuen Klassen von c-di-GMP Effektoren konzentriert. Einige c-di-GMP Effektoren mit unterschiedlichen Bindungsmotiven wurden kürzlich beschrieben, wie die CRP / FNR-Protein-Familie Bcam1349 in Burkholderia cenocepacia oder der Transkriptionsregulator FleQ in P. aeruginosa 9,10. Zusätzlich wurden c-di-GMP-spezifische RNA-Schalter kürzlich identifiziert und gezeigt, um die Genexpression in einer c-di-GMP-abhängige Weise 11 steuern. Die c-di-GMP-Bindungsmotive von verschiedenen Effektoren nur schlecht konserviert machen bioinformatischen Vorhersagen solcher Proteine ​​schwierig. Um dieses Problem anzugehen, ein biochemisches Verfahren, das auf der Verwendung eines c-di-GMP spe basiert entwickelten wirsche Erfassung Verbindung mit der Massenspektrometrie 1,12,13 kombiniert. Wir haben vor kurzem entwickelt, eine neuartige dreiwertigen c-di-GMP-Capture Verbindung (CDG-CC, Abbildung 1) 1. Diese chemischen Gerüst besteht aus: 1) als Köder, um c-di-GMP erfassen bindenden Proteinen verwendet eine c-di-GMP-Einheit, 2) eine UV-photoaktivierbare reaktive Gruppe verwendet werden, um vernetzen CDG-CC zu den gebundenen Proteine ​​und 3) einem Biotin, um die aufgenommenen Proteine ​​unter Verwendung von Streptavidin-beschichteten magnetischen Kügelchen zu isolieren. Die CDG-CC kann verwendet werden, um direkt und spezifisch einzufangen c-di-GMP-Effektoren aus komplexen Mischung von Makromolekülen, wie Zell-Lysaten. Capture-Verbindung auf der Basis chemischer und Proteomik Ansätze wurde bereits berichtet Anwendbarkeit auf einen breiten Bereich von Organismen, wie zB Caulobacter crescentus, Salmonella enterica Serovar typhimurium und S. aeruginosa 1,14. In diesem methodischen Papier,bieten wir eine eingehende Beschreibung des CCMS Verfahren unter Verwendung von Extrakten von P. aeruginosa als ein Beispiel. Diese Studie stellt CCMS als eine leistungsfähige und vielseitiges Werkzeug, um biochemisch identifiziert neue Komponenten in kleinen Molekülen Signalgebung beteiligt.

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

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

Beachtliche Fortschritte wurden im letzten Jahrzehnt auf die Identifizierung und Charakterisierung von Enzymen bei der Synthese (diguanylate Cyclasen) und Abbau (Phosphodiesterasen) beteiligt der second messenger c-di-GMP hergestellt. Im Gegensatz dazu ist wenig Information über die molekularen Mechanismen und Zellkomponenten durch die dieses Signalmoleküls reguliert eine Vielfalt von zellulären Prozessen. Die meisten der bekannten Effektor-Proteine ​​gehören zur Familie PilZ oder degeneriert diguanylate Cyclasen oder Phosphodiesterasen, die sich auf die Katalyse gegeben haben und Effektor-Funktion übernommen. So, um besser die zelluläre c-di-GMP-Netzwerk in einem weiten Bereich von Bakterien experimentelle Methodik zur Identifizierung und Validierung von neuartigen Effektoren für die zuverlässige in silico Vorhersage nicht definieren. Wir haben vor kurzem eine neuartige Erfassung Verbindung Massenspektrometrie (CCMS) basierte Technologie als ein mächtiges Werkzeug, umbiochemisch zu identifizieren und zu charakterisieren, c-di-GMP-bindende Proteine. Diese Technik wurde bereits berichtet anwendbar auf einen weiten Bereich von Organismen 1 sein. Hier geben wir eine ausführliche Beschreibung des Protokolls, die wir verwenden, um solche Signalkomponenten zu untersuchen. Als ein Beispiel verwenden wir P. aeruginosa, ein opportunistisches Pathogen in dem c-di-GMP spielt eine kritische Rolle bei der Virulenz und Biofilmkontrolle. CCMS identifizierten 74% (38/51) der bekannten oder vorhergesagten Komponenten des c-di-GMP-Netzwerk. Diese Studie erklärt die CCMS-Verfahren im Detail und stellt es als ein leistungsfähiges und vielseitiges Werkzeug, um neue Komponenten in kleinen Molekülen Signalisierung beteiligt sind.

Um neue c-di-GMP-Effektoren in P. identifizieren aeruginosa wir systematisch genutzt CCMS die löslichen und Membranfraktionen P. analysieren aeruginosa Stamm PAO1 aus einer log-Phase Kultur (OD 600 = 0,5). Hier werden wir zusammenfassen und diskutieren Vertreter Ergebnisse dieser Angelausflug. Vier unabhängige biologischen Replikate wurden verwendet. Für jeden Versuch zwei verschiedenen CDG-CC-Konzentrationen wurden verwendet (5 uM und 10 uM). Um die Spezifität zu unter...

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

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

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Capture Compound Mass Spectrometry - Ein leistungsfähiges Tool zum Identifizieren Novel c-di-GMP Effektorproteinen

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

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

Research

JoVE Journal

Biology

11.5K Aufrufe.  Biozentrum of the University of Basel. The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

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

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

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

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

Proteomics, um Proteine ​​interagieren mit P2X2 Ligand-Gated Kationenkanäle Identifizieren

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

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

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

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

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 ...

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

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

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

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

Split-Ubiquitin Basierend Membrane Yeast Two-Hybrid (Mythos) System: Ein wirksames Instrument zur Ermittlung von Protein-Protein-Wechselwirkungen

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

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Analyse großer Proteinkomplexe von Structural-Massenspektrometrie

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

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

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

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

PAR-Clip - Ein Verfahren zur Transkriptom-weiten die Bindungsstellen des RNA-bindende Proteine ​​Identifizieren

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

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Profiling von Methyltransferasen und andere S-Adenosyl- L-Homocystein-bindende Proteine ​​von 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 ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

Quantifizierung von Proteinen über ein Peptid Immunoaffinity Enrichment gekoppelt mit der Massenspektrometrie

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

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

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

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

Amid Wasserstoff / Deuterium-Austausch &amp; MALDI-TOF-Massenspektrometrie Analyse der PAK2 Activation

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

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Thiolredox Proteome Mit Isotopen-Massenspektrometrie Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...