Name: lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism
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Ethikerklärung: Alle Protokolle, die die Behandlung von menschlichen Proben über folgen den Richtlinien der University of Pennsylvania des Komitees Forschung mit menschlichen Ethik. 1. Herstellung von Puffern und 100x Stammlösungen <ol><li> Bereiten Sie 1 l Basis Tyrode-Puffer. Kombinieren 8,123 g NaCl, 1,428 g NaHCO 3, 0,466 g CaCl 2 ∙ 2 H 2 O, 0,224 g KCl und 0,095 g MgCl 2. Stellen Sie ein Gesamtvolumen von 1 l mit ddH 2 O. Fil...

<ol>
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Hier haben wir die Nützlichkeit von isolierten Blutplättchen als Plattform gezeigt für das Studium gestörte mitochondriale Stoffwechsel. Insbesondere haben wir metabolische Anpassung in Antwort auf komplexe I Hemmung durch Rotenon gekennzeichnet. Die vorliegende Studie wurde erweitert zuvor berichteten Ergebnisse über die Rolle der Komplex I-Hemmung durch Rotenon in Zelllinien menschlichen Blutplättchen. Wichtig ist, dass dies zeigte, dass Rotenon auch inhibierte Plättchen Succinyl-Co...

Dysfunctional mitochondrialen Stoffwechsel wurde in einer Vielzahl von Krankheiten , einschließlich Neurodegeneration, Krebs und Herz - Kreislauferkrankungen 30 gebracht. Als solche große Anstrengungen unternommen, auf die Charakterisierung metabolische Defekte wurden platziert, die Pathogenese der Erkrankung beitragen. Flüssigchromatographie-Tandem - Massenspektrometrie (LC-MS / MS) ist der Goldstandard für die Quantifizierung von Analyten aus komplexen biologischen Matrizen betrachtet und wird häufig für metabolische Studien beschäftigt 8. Doch wie es oft der Fall mit biomedizinischen Studien, eine leicht zugängliche und gut definierte Modell relevant für die menschliche Krankheit zu erreichen, ist eine Herausforderung. Viele Studien beschäftigen für die Erforschung der Wirkung von Xenobiotika oder genetische Anomalien auf den Zellstoffwechsel 7,9 zellulären Modellen umgewandelt. Die metabolische Neuprogrammierung , die in Krebszellen auftritt , kann confounding einzuführen Faktoren 21 und sind daher nicht ideal. Diese Probleme können sein circumvented mit primären Zellmodellen, obwohl sich eine ausreichende Biomasse für metabolische Analysen kann eine Herausforderung sein. Darüber hinaus verwendet die Auswirkungen der hohen Mengen an Antibiotika in der Kultur wurde als potentiell verwirrende mitochondrialen Studien 16 hervorgehoben. Menschliche Blutplättchen die Chance zu einer primären Zellmodell mit ausreichender mitochondrialen Inhalt für metabolische Studien 5,22,27,32 zu nutzen. Erstens können Blutplättchen leicht erworben werden, durch Blut von einzelnen Spendern anfertigt oder in großen Mengen von Blutbanken und bieten daher ein Modell, in dem externe Faktoren leicht gesteuert werden. Zweitens, die aufgrund ihrer geringen Größe, Plättchen kann leicht von anderen Blutbestandteilen mit minimaler Vorbereitungsarbeiten auch nur minimal ausgestatteten Labors 5 isoliert werden. unabhängig von der Transkriptionsregulation der Anmerkung, Blutplättchen enthalten keine Kerne und kann daher verwendet werden, Änderungen an den Stoffwechsel zu studieren. Hier zeigen wir, dassZusätzlich zur relativen Quantifizierung der Acyl-Coenzym A (CoA) Thioester kann das isolierte Thrombozyten-System verwendet werden Kohlenstoffmetabolismus zu untersuchen. Insbesondere stellen wir die Verwendung von metabolischer Markierung mit stabilen Isotopen (nicht radioaktiv) markiert [13 C 6] -Glucose und [13 C 16] -Palmitat Sonde Einbau des [13 C] -markierten in den wichtigen Metaboliten acetyl- CoA durch Oxidation Glykolyse oder Fettsäure. Dies bietet eine leistungsstarke, verallgemeinerbar und vielseitige Plattform auf Grund der umfangreichen Beteiligung von Acyl-CoA - Spezies in biochemischen Wege 13,24 und die Lenkbarkeit dieses Systems zu testen anderen Variablen, wie die Hemmung der Komplex I mit Rotenon 3,33. Zusätzlich zu den Informationen im Protokoll unten vorgesehen ist , eine ausführliche Beschreibung der Methoden zur Isotopenmarkierung verwendet und für die LC-MS-basierte Analysen in Basu und Blair 4.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Reagent</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Chloride (NaCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">746398</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Sodium Bicarbonate (NaHCO3)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">S5761</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Calcium Chloride Dihydrate (CaCl2 * H2O)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">223506</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Potassium Chloride (KCl)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">P9541</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">208337</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">G8270</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C6-Glucose</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td>389374</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Palmitic acid</td>
			<td style="width:92px;">Cayman</td>
			<td style="width:68px;">10006627</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">13C16-Palmitic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">605573</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Rotenone</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">R8875</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Trichloro Acetic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">T6399</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">5-Sulfosalicylic Acid</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">390275</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Acetonitirle</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A996-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Water (H2O)</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">W7-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Formic acid</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">85171</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Dimethyl Sulfoxide</td>
			<td style="width:92px;">Sigma-Aldrich</td>
			<td style="width:68px;">472301</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ethanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">04-355-222</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Methanol</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A454-4</td>
			<td style="width:85px;">(optima)</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Ammonium Acetate</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">A639-500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">2 mL Eppendorf Tubes</td>
			<td style="width:92px;">BioExpress</td>
			<td style="width:68px;">C-3229-1</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">LC vials (plastic)</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">186002640</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">10 mL Glass Centrifuge Tubes</td>
			<td style="width:92px;">Kimble Chase</td>
			<td style="width:68px;">73785-10</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Oasis Solid Phase Extraxtion (SPE) Columns</td>
			<td style="width:92px;">Waters</td>
			<td style="width:68px;">WAT094225</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:161px;">Pastuer Pipets</td>
			<td style="width:92px;">Fischer Scientific</td>
			<td style="width:68px;">13-678-200</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Name</td>
			<td style="width:92px;">Company</td>
			<td style="width:68px;">Catalog Number</td>
			<td style="width:85px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Equipment</td>
			<td style="width:92px;"></td>
			<td style="width:68px;"></td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:161px;">CO2 Water-Jacketed Incubator</td>
			<td style="width:92px;">Nuaire</td>
			<td style="width:68px;">AutoFlow NU-8500</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">Triple Quadropole Mass Spectrometer</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Finnigan TSQ Quantum</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:161px;">HPLC</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">Dionex Ultimate 3000</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:161px;">Source</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:68px;">HESI II</td>
			<td style="width:85px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:161px;">HPLC Column</td>
			<td style="width:92px;">Phenomenex</td>
			<td style="width:68px;">Luna C18</td>
			<td style="width:85px;">3 &#956;m particle size, 200 mm x 2 mm</td>
		</tr>
	</tbody>
</table>

lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Zur Demonstration der Nützlichkeit dieser Methode wir die Verallgemeinerung der zuvor beschriebenen Ausgleichs metabolische Adaptation reproduziert aus der Exposition gegenüber Rotenon. Dieser Befund wurde zuvor in Zellkulturmodelle identifiziert und diese Untersuchung zielte darauf ab, zu testen, ob diese metabolische Verschiebung tritt auch in Blutplättchen, die kernlose und nicht anfällig für den gleichen experimentellen Artefakte wie Zellkultur sind. Diese Arbeit wurde mit 6-Tag...

Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Hier zeigen wir isolierten menschlichen Blutplättchen kann als zugänglich ex vivo - Modell verwendet werden metabolische Anpassungen in Antwort auf den Komplex I Rotenon Inhibitor zu studieren. Dieser Ansatz beschäftigt Isotopenverfolgung und relative Quantifizierung durch Flüssigchromatographie-Massenspektrometrie und kann auf eine Vielzahl von Studiendesigns angewendet werden.

LC-MS-Analyse von menschlichen Blutplättchen als Plattform für ein Studium Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

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SCIENTISTS IN ACTION

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Technik zur Untersuchung Gliederfüßer und Mikrobielle Gemeinschaften im Baum Tissues

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Forschung

Umwelt

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Transient Gene Expression in Tabak mit Gibson Versammlung und die Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Die Nutzung Chironomidae (Diptera) Oberflächenschwimm Pupal Exuviae als schnelle Bioassessment Protokoll für die Wasserkörper

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Modifizierung und Anwendung eines Laubbläser-vac für Feld Probenahme von Arthropoden

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Thermal Scanning Conductometry (TSC) as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

Thermal Scanning Conductometry TSC as a General Method for Studying and Controlling the Phase Behavior of Conductive Physical Gels

Thermische Conductometry (TSC) scannen, als eine allgemeine Methode für die Untersuchung und Kontrolle das Phasenverhalten von leitfähigen körperlichen Gele

The thermal scanning conductometry protocol is a new approach in studying ionic gels based on low molecular weight gelators. The method is designed to ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

Quantifizierung der Pflanze lösliche Protein und verdauliche Kohlenhydrate Inhalt, Verwendung von Mais (Zea Mays) als ein Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Untersuchung neurobehavioraler Auswirkungen von Umweltschadstoffen auf Zebrafischlarven

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

The Effect of Construction and Demolition Waste Plastic Fractions on Wood-Polymer Composite Properties

Die Auswirkungen von Bau- und Abbruchabfällen kunststofffraktioniert auf Holz-Polymer-Verbundeigenschaften

Construction and demolition waste (CDW), including valuable materials such as plastics, have a remarkable influence on the waste sector. In order for ...

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

Ein gnotobiotisches System zur Untersuchung der Mikrobiom-Montage in der Phyllosphäre und in der gemüseischen Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Mikroplot-Design und Pflanzen- und Bodenprobenvorbereitung für 15Stickstoffanalysen

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...