MH reconnaît le soutien de la «Deutsche Forschungsgemeinschaft» (DFG). Auteur contributions: MH conçu recherche; KT, JS et MT a effectué des recherches et a analysé les données; KT et MH a écrit le papier.

Les auteurs déclarent aucun intérêt financier concurrent.

Différentes études protéomiques quantitatives utilisant stable marquage isotopique ont été publiées dans les dernières années. Dans ces expériences, généralement de deux échantillons différents sont comparés, dont un échantillon est marqué avec un isotope stable. Par la suite, des protéines ou des peptides à partir des deux échantillons sont combinés dans un rapport égal, et en outre 48 traitées ensemble. Ces études ont l&#39;intention souvent comparer compartiments définis isolés cel...

processus de photosynthèse dans les membranes des thylakoïdes de plantes et d&#39;algues peuvent fonctionner en mode linéaire et cyclique. Au cours de l&#39;écoulement linéaire d&#39;électrons (LEF) de photosystème I (PSI), photosystème II (PSII) et le cytochrome b 6 / f finalement transférer des électrons de l&#39;eau à NADP + 1, conduisant à la génération de NADPH et ATP 2. En revanche, le flux d&#39;électrons cyclique (CEC), qui est connu pour être induit dans des conditions d&#39;environnement variées telles que des conditions d&#39;état 2 3 et anaérobies 4, les résultats de la re-réduction de l&#39;ISP oxydé par l&#39;injection d&#39;électrons dans la chaîne de transport d&#39;électrons. Ce processus peut avoir lieu soit sur ​​le côté du stroma de la cytochrome b 6 / f complexe 1 ou à la piscine de plastoquinone 5 et génère de l&#39;ATP, mais pas de la NADPH 2. L&#39;objectif du protocole est de démontrer présenté une spectrométrie de masse (MS) sur la base de méthode pour l&#39;analyse comparative, quantitative des complexes multiprotéiques dans les membranes des thylakoïdes de Chlamydomonas reinhardtii de mieux comprendre la composition de ces complexes dans des conditions différentes (illustrés en comparant différentes souches génétiquement). Cette approche a été appliquée dans une publication de Terashima et al., En 2012 montrant une Ca 2 + régulation dépendant de CEF dans C. reinhardtii médiée par un complexe multiprotéique comprenant les protéines CAS, ANR1, et PGRL1 6. La procédure sera expliquée par analyse comparative de la composition de la CEC-supercomplexe dans deux souches génétiquement différentes, profitant ainsi de l&#39;étiquetage une des deux souches avec de l&#39;azote lourd (15 N). Brièvement, le protocole comprend la préparation de membranes thylacoïdes, suivie d&#39;une solubilisation du détergent et le fractionnement de complexes photosynthétiques dans un gradient de densité de saccharose. Après fractionnement du gradient, choisi fractions de deux souches sont mélangées à base de volume égal, séparées par SDS-PAGE suivie d&#39;une digestion in-gel et l&#39;analyse quantitative subséquente de la sclérose en plaques. Comme mentionné ci-dessus, CEF est induite dans différentes conditions environnementales et une publication à partir de 2010 démontre l&#39;isolement d&#39;un CEF-supercomplexe fonctionnelle de l&#39;état 2 cellules verrouillées de C. reinhardtii 7, qui a été réalisée par des membranes de séparation thylacoïdiennes solubilisées sur un gradient de densité de sucrose pendant ultracentrifugation. Différent de Iwai et al. 7, le protocole présenté décrit l&#39;isolement de la CEC-supercomplexe cultivé en anaérobiose à partir de C. cultures reinhardtii en suivant une procédure alternative. Celui-ci comprend des changements dans le protocole d&#39;isolement thylacoïdes ainsi que les différences concernant l&#39;étape de solubilisation et la séparation des complexes protéiques par ultracentrifugation. Dans le présent protocole, les membranes des thylakoïdessont isolées en appliquant le mode opératoire publié par Chua et Bennoun 8, tandis que les tampons utilisés pour la préparation des thylakoïdes par Iwai et al. contenue 25 Mes mM, 0,33 M de saccharose, 5 mM de MgCl2, NaCl 1,5 mM (pH 6,5) comme décrit 9. La solubilisation a été effectuée avec de 0,7 à 0,8% de détergent (n-tridécyl-β-D-maltoside) pendant 30 min sur de la glace dans le cas d&#39;Iwai et collaborateurs, tandis que la méthode de solubilisation décrit ici repose sur l&#39;utilisation de 0,9% de détergent (n -dodécyl-β-D-maltoside (β-DM)) et est effectuée pour seulement 20 min sur la glace. Les deux groupes ont utilisé 0,8 mg de chlorophylle par ml pour la solubilisation avec le détergent respectif. Pour la séparation de complexes à partir de membranes photosynthétiques des thylacoïdes solubilisées Iwai et al. Appliqué entre les concentrations de saccharose de 0,1 à 1,3 M, tandis que les auteurs de ce protocole utilise des concentrations allant de 0,4 à 1,3 M. La dernière différence est la vitesse de centrifugation, ce qui est faible par rapport à l&#39;eapublication rlier. Solubilisation des membranes thylacoïdes avec des détergents non ioniques suivie d&#39;un fractionnement gradient de densité de saccharose a déjà été utilisée dans de nombreuses études, allant des années 1980 jusqu&#39;à aujourd&#39;hui, 7, 9 à 14 et également à l&#39;application de marquage métabolique des protéines est un procédé très répandu dans le domaine de la protéomique. La méthode décrite s&#39;applique à l&#39;étiquetage 15 N métabolique pour l&#39;une des deux souches comparées par la mise en culture en présence d&#39;azote lourd comme seule source d&#39;azote sous la forme de 15 N NH 4 Cl, qui est incorporée dans tous les acides aminés conduisant à une masse décaler en fonction de la séquence d&#39;acides aminés du peptide. Lors de l&#39;analyse d&#39;un mélange de 14 N et 15 N à l&#39;intérieur une MS exécuté, ce changement de masse peut être utilisée pour déterminer l&#39;origine de l&#39;échantillon pour chaque peptide et le peptide parent abondances peuvent être calculées représentant les abondances relatives pour l&#39;correspondentment 15 protéine. De nombreuses études protéomiques quantitatives sur C. reinhardtii sont disponibles, qui permettent de comparer une quantité définie de protéines pour analyser les changements dans le protéome entre les conditions expérimentales (par exemple, les changements dans le protéome en raison de nutriments 16-19 ou lumière le stress 20,21). Par rapport à ces études, l&#39;approche a présenté des volumes égaux d&#39;échantillons sont combinés et analysés. Cette configuration permet d&#39;étudier le comportement de migration des protéines dans le gradient et en outre à analyser la composition des différents complexes en ce qui concerne les souches étudiées. Cette méthode sera expliquée en se concentrant principalement sur ​​trois protéines: Le premier candidat est la protéine de capteur calcium CAS chloroplastique-localisé, qui a été montré être impliqué dans le photo-acclimatation en C. reinhardtii 22. Le calcium est considéré comme un signal importantment ion pour les voies qui sont activés en raison de différentes contraintes biotiques et abiotiques conduisant finalement à des changements dans l&#39;expression des gènes et la physiologie des cellules 23 et il a été proposé que les chloroplastes pourraient contribuer à Ca 2 + cellulaire signalisation via la protéine CAS 22,24,25. La seconde protéine est ANR1 (réponse anaérobie 1 6), une protéine qui a été montré être induite dans des conditions anoxiques dans la croissance de C. reinhardtii 26. Notamment, CAS ainsi que ANR1 ont été identifiés comme sous-unités du CEF-supercomplexe et ailleurs, en utilisant des approches de génétique inverse, il a été démontré que les deux protéines contribuent fonctionnellement au CEF in vivo 6, en soutenant leur rôle en tant que sous-unités fonctionnelles de ce complexe protéique. La troisième protéine est la protéine thylakoid PGR5-Like 1 (PGRL1), qui a été montré pour être impliqués dans le CECR dans Chlamydomonas 4,27 ainsi que chez Arabidopsis 5,28 et était aussi identified dans le travail de Iwai et al. 7 Cette approche sera présentée en montrant les résultats de deux expériences différentes: type sauvage (WT) versus (vs) une souche ΔPSI 29, présentant une délétion du gène du CCSP, codant pour une sous-unité du photosystème essentiel je, qui fait également partie de la CEF-supercomplexe et WT vs un pgrl1 knock-out souche 4. Pour chacune de ces expériences, la composition quantitative de la CEC-supercomplexe entre un N-15 et une souche 14 N-marqué a été comparé.

<table border="0" cellpadding="0" cellspacing="0" width="1135">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Chemicals</td>
			<td style="width:427px;"></td>
			<td style="width:119px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Acetic acid</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0662</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Acetone</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2300</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Acetonitrile Optigrade f&#252;r LC-MS</td>
			<td style="width:427px;">Diagonal 
			website: https://www.diagonal.de/</td>
			<td style="width:119px;">9340</td>
			<td style="width:199px;">harmful, work with gloves 
			see protocol text for further precautions</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Ammonium chloride 15N</td>
			<td style="width:427px;">Cambridge Isotope Laboratories 
			website: http://www.isotope.com/cil/index.cfm</td>
			<td style="width:119px;">39466-62-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ammonium chloride 14N</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0988</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ammonium hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ammonium sulfate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3598</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Coomassie brilliant blue R-250</td>
			<td style="width:427px;">Fisher Scientific 
			website: http://www.de.fishersci.com/index.php/deindex</td>
			<td>10041653</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">n-Dodecyl-&#946;-D-maltoside</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0819</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">EDTA</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2937</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Formic acid&#160;&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3858</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hepes</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3724</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Magnesium chloride</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td>A4425</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Methanol</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2954</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Phosphorous acid</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A0989</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Di-Potassium hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1042</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Potassium di-hydrogenphosphate&#160;</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1043</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sodium hydroxide</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1551</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Sucrose</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A1125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tricine</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A3954</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Tris</td>
			<td style="width:427px;">AppliChem 
			webiste: http://www.applichem.com/home/</td>
			<td style="width:119px;">A2264</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Trypsin (sequencing grade modified) and Trypsin buffer</td>
			<td style="width:427px;">Promega 
			website: http://www.promega.de/</td>
			<td style="width:119px;">V5111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;"></td>
			<td style="width:427px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Equipment&#160;</td>
			<td style="width:427px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Neboulizer (BioNeb cell disruptor)</td>
			<td style="width:427px;">Glas-Col 
			website: http://www.glascol.com/product/subproduct/id/75</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Centrifuge tubes (14 x 89 mm)&#160; 
			for preparation of takahashi style gradients</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;">331372</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Centrifuge tubes 25 x 89 mm 
			for preparation of thylakoid isolation gradients&#160;</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;">344058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Coulter Avanti Centrifuge J-20 XP</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fuchs-Rosenthal cell couting chamber</td>
			<td style="width:427px;">Diagonal 
			website: https://www.diagonal.de/</td>
			<td style="width:119px;">449/72</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Homogenizer (Potter) 50 ml&#160;</td>
			<td style="width:427px;">Fisherbrand 
			website: http://www.de.fishersci.com/index.php/defisherbrand</td>
			<td style="width:119px;">10618242</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Pistil for homogenizer</td>
			<td style="width:427px;">Fisherbrand 
			website: http://www.de.fishersci.com/index.php/defisherbrand</td>
			<td style="width:119px;">105252220</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Ultracentrifuge (Optima XPN-80 Ultracentrifuge)</td>
			<td style="width:427px;">Beckman Coulter 
			website: http://www.beckmancoulter.de/</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;"></td>
			<td style="width:427px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Other</td>
			<td style="width:427px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:392px;">Antibodies&#160;</td>
			<td style="width:427px;">Agrisera 
			website: http://www.agrisera.com/en/index.html</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a new approach for the comparative analysis of multiprotein complexes based on 15n metabolic labeling and quantitative mass spectrometry

Le protocole mis en place un outil pour l&#39;analyse des complexes multiprotéiques dans la membrane des thylakoïdes, en révélant un aperçu de la composition complexe dans des conditions différentes. Dans ce protocole, l&#39;approche est démontrée en comparant la composition de la protéine complexe responsable de flux d&#39;électrons cyclique (CEF) dans Chlamydomonas reinhardtii, isolé à partir de différentes souches génétiquement. Le procédé comprend l&#39;isolement de membranes thylacoïdes, suivi de leur séparation dans des complexes multiprotéiques par le saccharose centrifugation à gradient de densité, SDS-PAGE, immunodétection et comparative, la spectrométrie de masse quantitative (MS) sur la base de marquage métabolique différentiel (14 N / 15 N) de l&#39; souches analysées. Membranes thylacoïdes solubilisées détergents sont chargés sur des gradients de densité de saccharose à une concentration de chlorophylle égal. Après ultracentrifugation, les gradients sont séparées en fractions, qui sont analysés en masse-spectrometry basé sur le volume égal. Cette approche permet l&#39;étude de la composition dans les fractions de gradient, les analyser le comportement de migration des protéines différentes, notamment en se concentrant sur ANR1, CAS, et PGRL1. De plus, cette méthode est démontrée en confirmant les résultats immuno et, en outre, en soutenant les conclusions des études précédentes (l&#39;identification et la migration de PSI-dépendante des protéines qui ont été précédemment décrits comme faisant partie de la FEC-supercomplexe comme PGRL1, FNR, et cyt f). Notamment, cette approche est applicable à traiter un large éventail de questions pour lesquelles ce protocole peut être adopté et utilisé par exemple pour des analyses comparatives de composition complexe multiprotéique isolé des conditions environnementales distinctes.

L&#39;approche de protéomique quantitative introduit vise à caractériser la composition de complexes multiprotéiques dans les membranes des thylakoïdes démontré par l&#39;analyse comparative des composants CEF-supercomplexe dans génétiquement différente C. reinhardtii souches. La méthode décrite a été appliquée avec succès par Terashima et al. 6 et comprend l&#39;isolement des membranes thylacoïdes de anaérobies grandi cultures, suivis par solubilisation avec un détergent....

Watch this Scientific Journal Video about A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry at JoVE.com

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

L&#39;approche décrite comparative, protéomique quantitative vise à obtenir un aperçu de la composition de complexes multiprotéiques dans des conditions différentes et est démontrée en comparant différentes souches génétiquement. Pour l&#39;analyse quantitative des volumes égaux de fractions différentes d&#39;un gradient de densité de saccharose sont mélangés et analysés par spectrométrie de masse.

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 ...

a-new-approach-for-comparative-analysis-multiprotein-complexes-based

Research

JoVE Journal

Biology

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

12.1K vues.  University of Münster.  L'approche décrite comparative, protéomique quantitative vise à obtenir un aperçu de la composition de complexes multiprotéiques dans des conditions différentes et est démontrée en comparant différentes souches génétiquement. Pour l'analyse quantitative des volumes égaux de fractions différentes d'un gradient de densité de saccharose sont mélangés et analysés par spectrométrie de masse. 

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

MALDI Sample Preparation: the Ultra Thin Layer Method

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

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

Préparation des échantillons MALDI: la méthode de la couche ultra mince

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

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 ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

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

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

L&#39;analyse des complexes protéiques par spectrométrie de masse Grande structurels

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

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Bleu d&#39;électrophorèse sur gel de polyacrylamide natif (BN-PAGE) pour l&#39;analyse des complexes multiprotéiques de lysats cellulaires

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

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

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

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

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

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

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

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

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

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

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 ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Marquage métabolique et la membrane de fractionnement pour comparative Analyse protéomique de<em&gt; Arabidopsis thaliana</em&gt; Cultures Suspension cellulaires

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

L&#39;approche ChroP Combine puce et la spectrométrie de masse à Disséquer protéomiques paysages Locus spécifiques de la chromatine

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Une nouvelle technique pour l&#39;analyse quantitative de la perte de cheveux chez la souris Utilisation de l&#39;analyse des niveaux de gris

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

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 ...