Name: lc-ms analysis of human platelets as a platform for studying mitochondrial metabolism
Uploaded: 2016-04-04T15:00:00
Description: Watch this Scientific Journal Video about LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism at JoVE.com

Noi riconosciamo il sostegno del NIH sovvenzioni P30ES013508 e T32ES019851.

Etica Dichiarazione: Tutti i protocolli in materia di trattamento dei campioni umani seguono le linee guida del comitato di etica della ricerca human L&#39;Università della Pennsylvania di. 1. Preparazione di buffer e 100x archivio Solutions <ol><li> Preparare 1 L di tampone di base di Tyrode. Unire 8,123 g di NaCl, 1.428 g NaHCO 3, 0,466 g CaCl 2 ∙ 2 H 2 O, 0,224 g KCl, e 0,095 g MgCl 2. Regolare il volume totale a 1 L con DDH 2 O....

<ol>
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Qui abbiamo dimostrato l&#39;utilità delle piastrine isolate come piattaforma per lo studio del metabolismo mitocondriale perturbato. In particolare, abbiamo caratterizzato adattamento metabolico in risposta alla inibizione del complesso I da rotenone. Il presente studio ha esteso risultati precedentemente segnalati sul ruolo di inibizione del complesso da rotenone in linee cellulari di piastrine umane. È importante sottolineare che questo ha rivelato che la formazione delle piastrine rote...

Metabolismo mitocondriale disfunzionale è stata implicata in una vasta gamma di malattie, tra cui neurodegenerazione, il cancro, le malattie cardiovascolari e 30. Come tale, grande sforzo è stato posto sulla caratterizzazione difetti metabolici che contribuiscono alla patogenesi della malattia. Spettrometria di massa cromatografia liquida-tandem (LC-MS / MS) è considerata il gold standard per la quantificazione di analiti da matrici biologiche complesse ed è spesso impiegato per studi metabolici 8. Tuttavia, come è spesso il caso con studi biomedici, raggiungendo un modello accessibile e ben definito rilevante per la malattia umana è una sfida. Molti studi impiegano trasformate modelli cellulari per sondare l&#39;impatto di xenobiotici o anomalie genetiche sul metabolismo cellulare 7,9. La riprogrammazione metabolica che si verifica nelle cellule tumorali può introdurre confusione fattori di 21 e non sono quindi l&#39;ideale. Questi problemi possono essere circumvented con modelli cellulari primarie, anche se l&#39;ottenimento di biomassa sufficiente per le analisi del metabolismo può essere impegnativo. Inoltre, l&#39;impatto di elevate quantità di antibiotici utilizzati in cultura è stato evidenziato come potenzialmente confondenti studi mitocondriali 16. Piastrine umane permettersi l&#39;opportunità di utilizzare un modello di cellula primaria con sufficiente contenuto mitocondriale per studi metabolici 5,22,27,32. Innanzitutto, le piastrine possono essere facilmente acquisite, attraverso il sangue trae da donatori individuali o in grandi volumi da banche del sangue, e quindi fornire un modello in cui fattori esterni possono essere facilmente controllati. In secondo luogo, a causa delle loro piccole dimensioni, le piastrine possono essere facilmente isolati da altri componenti del sangue con il lavoro preparatorio minima in laboratori attrezzati minimamente 5. Da segnalare, le piastrine non contengono nuclei e possono quindi essere utilizzati per studiare le alterazioni al metabolismo indipendentemente dalla regolazione trascrizionale. Qui mostriamo cheoltre alla quantificazione relativa di (CoA) tioesteri acil-coenzima A, il sistema piastrinico isolato può essere utilizzato per esaminare il metabolismo carbonio. In particolare, si segnala l&#39;utilizzo di marcatura metabolica con isotopi stabili (non radioattivo) marcato [13 C 6] Glucosio e [13 C 16] -palmitate per sondare incorporazione della [13 C] -label nella importante acetil metabolita CoA via glicolisi o ossidazione degli acidi grassi. Questo fornisce una piattaforma potente, generalizzabile, e versatile grazie alla massiccia partecipazione di specie acil-CoA in vie biochimiche 13,24 e la trattabilità di questo sistema per testare altre variabili, come ad esempio l&#39;inibizione del complesso con rotenone 3,33. Oltre alle informazioni fornite nel protocollo di seguito, una descrizione dettagliata dei metodi utilizzati per l&#39;etichettatura degli isotopi e per le analisi LC-MS-based può essere trovato in Basu e 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.

Per dimostrare l&#39;utilità di questa metodologia abbiamo riprodotto la generalizzabilità dei precedentemente descritto adattamento metabolico compensativo esposizione al rotenone. Questo risultato è stato precedentemente identificato in modelli di coltura cellulare e questa indagine aveva lo scopo di verificare se questo cambiamento metabolico si verifica anche nelle piastrine, che sono anuclear e non incline agli stessi artefatti sperimentali come coltura cellulare. Questo lavoro ?...

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

Qui mostriamo isolato piastrine umane possono essere utilizzati come accessibili ex vivo modello per studiare adattamenti metabolici in risposta al complesso I inibitore rotenone. Questo approccio impiega tracing isotopica e relativa quantificazione dal liquido spettrometria cromatografia massa e può essere applicato ad una varietà di disegni di studio.

LC-MS Analisi delle piastrine umane come una piattaforma per lo Studio del metabolismo mitocondriale

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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Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Tecnica per lo Studio artropodi e comunità microbiche all&#39;interno Albero tessuti

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 ...

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

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

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

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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

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Quantificare la pianta proteina solubile e digeribile carboidrati contenuti, utilizzando mais (Zea mays) As an 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

Studiare gli effetti neurocomportamentali degli inquinanti ambientali sulle larve dei pesci zebra

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

L'effetto delle frazioni di plastica dei rifiuti di costruzione e demolizione sulle proprietà composite wood-Polymer

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

Un sistema gnotobiotico per studiare l'assemblaggio del microbioma nella Phyllosphere e nella fermentazione vegetale

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

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Progettazione di microplot e preparazione di campioni di piante e suolo per 15Analisi dell'azoto

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. ...