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

Agradecemos o apoio do NIH subvenções P30ES013508 e T32ES019851.

Declaração de Ética: Todos os protocolos relativos ao tratamento de amostras humanas seguir as orientações do comitê de ética em pesquisa humana da Universidade da Pensilvânia. 1. Preparação de tampões e soluções de reserva 100X <ol><li> Prepare 1 litro de solução tampão de Tyrode de base. Combinar 8,123 g de NaCl, 1,428 g de NaHCO3, 0,466 g de CaCl 2 ∙ 2 H 2 O, 0,224 g de KCl, e 0,095 g de MgCl 2. Ajustar o volume total a 1 L c...

<ol>
	<li>Ault, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+utility+of+flow+cytometry+in+the+study+of+platelets.">The clinical utility of flow cytometry in the study of platelets.</a> Semin. Hematol. 38 (2), 160-168 (2001).</li><li>Avila, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+mitochondrial+dysfunction+is+evident+in+type+2+diabetes+in+association+with+modifications+of+mitochondrial+anti-oxidant+stress+proteins.">Platelet mitochondrial dysfunction is evident in type 2 diabetes in association with modifications of mitochondrial anti-oxidant stress proteins.</a> Exp. Clin. Endocrinol. Diabetes. 120 (4), 248-251 (2012).</li><li>Basu, S. S., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rotenone-mediated+changes+in+intracellular+coenzyme+A+thioester+levels:+implications+for+mitochondrial+dysfunction.">Rotenone-mediated changes in intracellular coenzyme A thioester levels: implications for mitochondrial dysfunction.</a> Chem. Res. Toxicol. 24 (10), 1630-1632 (2011).</li><li>Basu, S. S., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SILEC:+a+protocol+for+generating+and+using+isotopically+labeled+coenzyme+A+mass+spectrometry+standards.">SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards.</a> Nat. Protoc. 7 (1), 1-12 (2012).</li><li>Basu, S. S., Deutsch, E. C., Schmaier, A. A., Lynch, D. R., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelets+as+a+platform+to+monitor+metabolic+biomarkers+using+stable+isotopes+and+LC-MS.">Human platelets as a platform to monitor metabolic biomarkers using stable isotopes and LC-MS.</a> Bioanalysis. 5 (24), 3009-3021 (2013).</li><li>Berson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+experimental+buprenorphine+hepatotoxicity:+major+role+of+mitochondrial+dysfunction+versus+metabolic+activation.">Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.</a> J. Hepatol. 34 (2), 261-269 (2001).</li><li>Castell, J. V., Jover, R., Martinez-Jimenez, C. P., Gomez-Lechon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+cell+lines:+their+use,+scope+and+limitations+in+drug+metabolism+studies.">Hepatocyte cell lines: their use, scope and limitations in drug metabolism studies.</a> Expert. Opin. Drug Metab Toxicol. 2 (2), 183-212 (2006).</li><li>Ciccimaro, E., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable-isotope+dilution+LC-MS+for+quantitative+biomarker+analysis.">Stable-isotope dilution LC-MS for quantitative biomarker analysis.</a> Bioanalysis. 2 (2), 311-341 (2010).</li><li>Dang, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Links+between+metabolism+and+cancer.">Links between metabolism and cancer.</a> Genes Dev. 26 (9), 877-890 (2012).</li><li>Darnell, M., Weidolf, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+xenobiotic+carboxylic+acids:+focus+on+coenzyme+A+conjugation,+reactivity,+and+interference+with+lipid+metabolism.">Metabolism of xenobiotic carboxylic acids: focus on coenzyme A conjugation, reactivity, and interference with lipid metabolism.</a> Chem. Res. Toxicol. 26 (8), 1139-1155 (2013).</li><li>Des Rosiers, C., Fernandez, C. A., David, F., Brunengraber, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversibility+of+the+mitochondrial+isocitrate+dehydrogenase+reaction+in+the+perfused+rat+liver.+Evidence+from+isotopomer+analysis+of+citric+acid+cycle+intermediates.">Reversibility of the mitochondrial isocitrate dehydrogenase reaction in the perfused rat liver. Evidence from isotopomer analysis of citric acid cycle intermediates.</a> J. Biol. Chem. 269 (44), 27179-27182 (1994).</li><li>Ellis, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling+detects+early+effects+of+environmental+and+lifestyle+exposure+to+cadmium+in+a+human+population.">Metabolic profiling detects early effects of environmental and lifestyle exposure to cadmium in a human population.</a> BMC. Med. 10, 61 (2012).</li><li>Grevengoed, T. J., Klett, E. L., Coleman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyl-CoA+metabolism+and+partitioning.">Acyl-CoA metabolism and partitioning.</a> Annu. Rev. Nutr. 34, 1-30 (2014).</li><li>Haynes, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+fatty+acyl-coenzyme+As+in+mammalian+cells+by+liquid+chromatography-electrospray+ionization+tandem+mass+spectrometry.">Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry.</a> J. Lipid Res. 49 (5), 1113-1125 (2008).</li><li>Ikegawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cholyl-adenylate+in+rat+liver+microsomes+by+liquid+chromatography/electrospray+ionization-mass+spectrometry.">Characterization of cholyl-adenylate in rat liver microsomes by liquid chromatography/electrospray ionization-mass spectrometry.</a> Anal. Biochem. 266 (1), 125-132 (1999).</li><li>Kalghatgi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bactericidal+antibiotics+induce+mitochondrial+dysfunction+and+oxidative+damage+in+Mammalian+cells.">Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in Mammalian cells.</a> Sci. Transl. Med. 5 (192), 192ra85 (2013).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Metabolomics+with+Acyl-CoA+Profiling+Reveals+Widespread+Remodeling+in+Response+to+Diet.">High-Resolution Metabolomics with Acyl-CoA Profiling Reveals Widespread Remodeling in Response to Diet.</a> Mol. Cell Proteomics. 14 (6), 1489-1500 (2015).</li><li>Lopez-Gallardo, E., Iceta, R., Iglesias, E., Montoya, J., Ruiz-Pesini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OXPHOS+toxicogenomics+and+Parkinson's+disease.">OXPHOS toxicogenomics and Parkinson's disease.</a> Mutat. Res. 728 (3), 98-106 (2011).</li><li>Magnes, C., Sinner, F. M., Regittnig, W., Pieber, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC/MS/MS+method+for+quantitative+determination+of+long-chain+fatty+acyl-CoAs.">LC/MS/MS method for quantitative determination of long-chain fatty acyl-CoAs.</a> Anal. Chem. 77 (9), 2889-2894 (2005).</li><li>Mauriala, T., Herzig, K. H., Heinonen, M., Idziak, J., Auriola, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+long-chain+fatty+acid+acyl-coenzyme+A+compounds+using+liquid+chromatography-electrospray+ionization+tandem+mass+spectrometry.">Determination of long-chain fatty acid acyl-coenzyme A compounds using liquid chromatography-electrospray ionization tandem mass spectrometry.</a> J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 808 (2), 263-268 (2004).</li><li>Murphy, T. A., Dang, C. V., Young, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopically+nonstationary+13C+flux+analysis+of+Myc-induced+metabolic+reprogramming+in+B-cells.">Isotopically nonstationary 13C flux analysis of Myc-induced metabolic reprogramming in B-cells.</a> Metab Eng. 15, 206-217 (2013).</li><li>Paglia, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+analysis+of+platelets+during+storage:+a+comparison+between+apheresis-+and+buffy+coat-derived+platelet+concentrates.">Metabolomic analysis of platelets during storage: a comparison between apheresis- and buffy coat-derived platelet concentrates.</a> Transfusion. 55 (2), 301-313 (2015).</li><li>Patti, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+strategies+for+untargeted+metabolomics.">Separation strategies for untargeted metabolomics.</a> J. Sep. Sci. 34 (24), 3460-3469 (2011).</li><li>Robishaw, J. D., Neely, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coenzyme+A+metabolism.">Coenzyme A metabolism.</a> Am. J. Physiol. 248 (1 Pt 1), E1-E9 (1985).</li><li>Rodgers, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+platelet+physiology+and+laboratory+evaluation+of+platelet+function.">Overview of platelet physiology and laboratory evaluation of platelet function.</a> Clin. Obstet. Gynecol. 42 (2), 349-359 (1999).</li><li>Salles, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+high-throughput+ELISA+assay+for+platelet+function+testing+using+platelet-rich+plasma+or+whole+blood.">Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.</a> Thromb. Haemost. 104 (2), 392-401 (2010).</li><li>Snyder, N. W., Basu, S. S., Worth, A. J., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+propionic+acid+to+a+novel+acyl-coenzyme+A+thioester+by+mammalian+cell+lines+and+platelets.">Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets.</a> J. Lipid Res. 56 (1), 142-150 (2015).</li><li>Snyder, N. W., Basu, S. S., Zhou, Z., Worth, A. J., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+dilution+liquid+chromatography/mass+spectrometry+analysis+of+cellular+and+tissue+medium-+and+long-chain+acyl-coenzyme+A+thioesters.">Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters.</a> Rapid Commun. Mass Spectrom. 28 (16), 1840-1848 (2014).</li><li>Snyder, N. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+stable+isotope-labeled+acyl-coenzyme+A+thioesters+by+yeast+stable+isotope+labeling+by+essential+nutrients+in+cell+culture.">Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture.</a> Anal. Biochem. 474, 59-65 (2015).</li><li>Wallace, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+diseases+in+man+and+mouse.">Mitochondrial diseases in man and mouse.</a> Science. 283 (5407), 1482-1488 (1999).</li><li>Wojtovich, A. P., Brookes, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+II+inhibitor+atpenin+A5+protects+against+cardiac+ischemia-reperfusion+injury+via+activation+of+mitochondrial+KATP+channels.">The complex II inhibitor atpenin A5 protects against cardiac ischemia-reperfusion injury via activation of mitochondrial KATP channels.</a> Basic Res. Cardiol. 104 (2), 121-129 (2009).</li><li>Worth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotopes+and+LC-MS+for+monitoring+metabolic+disturbances+in+Friedreich's+ataxia+platelets.">Stable isotopes and LC-MS for monitoring metabolic disturbances in Friedreich's ataxia platelets.</a> Bioanalysis. 7 (15), 1843-1855 (2015).</li><li>Worth, A. J., Basu, S. S., Snyder, N. W., Mesaros, C., Blair, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neuronal+cell+mitochondrial+complex+I+with+rotenone+increases+lipid+beta-oxidation,+supporting+acetyl-coenzyme+A+levels.">Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid beta-oxidation, supporting acetyl-coenzyme A levels.</a> J. Biol. Chem. 289 (39), 26895-26903 (2014).</li><li>Zhou, L., Schmaier, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+testing+in+platelet-rich+plasma:+description+of+procedures+with+the+aim+to+develop+standards+in+the+field.">Platelet aggregation testing in platelet-rich plasma: description of procedures with the aim to develop standards in the field.</a> Am. J. Clin. Pathol. 123 (2), 172-183 (2005).</li></ol>

Aqui temos demonstrado a utilidade de plaquetas isoladas como uma plataforma para o estudo do metabolismo mitocondrial perturbado. Especificamente, temos caracterizado adaptação metabólica em resposta à inibição do complexo I por rotenona. O presente estudo se estendeu resultados reportados anteriormente sobre o papel da inibição do complexo I por rotenona em linhas celulares para plaquetas humanas. Importante, este revelou que a formação de rotenona também inibida de plaquetas su...

Metabolismo mitocondrial disfuncional tem sido implicada numa ampla variedade de doenças, incluindo a neurodegeneração, cancro, doenças cardiovasculares e 30. Como tal, um grande esforço foi colocado sobre a caracterização de defeitos metabólicos que contribuem para a patogénese da doença. Espectrometria de massa de cromatografia líquida-tandem (LC-MS / MS) é considerada o padrão-ouro para a quantificação de analitos a partir de matrizes biológicas complexas e muitas vezes é empregada para estudos metabólicos 8. No entanto, como é frequentemente o caso com estudos biomédicos, atingindo um modelo acessível e bem definida relevantes para doenças humanas é um desafio. Muitos estudos empregam transformado modelos celulares para sondar o impacto de xenobióticos ou anormalidades genéticas no metabolismo celular 7,9. A reprogramação metabólica que ocorre em células cancerosas podem introduzir factores de confusão 21 e, portanto, não são ideais. Estas questões podem ser circumvented com modelos de células primárias, embora a obtenção de biomassa suficiente para análises metabólicas pode ser um desafio. Além disso, o impacto de elevadas quantidades de antibióticos utilizados na cultura tem sido destacado como estudos mitocondriais potencialmente de confusão 16. Plaquetas humanas dar a oportunidade de utilizar um modelo de célula primária com conteúdo mitocondrial suficiente para estudos metabólicos 5,22,27,32. Em primeiro lugar, as plaquetas podem ser facilmente adquiridos, através de exames de sangue de doadores individuais, ou em grandes volumes de bancos de sangue e, portanto, fornecer um modelo em que fatores externos podem ser facilmente controlado. Em segundo lugar, devido ao seu pequeno tamanho, as plaquetas podem ser facilmente isolados a partir de outros componentes do sangue com o trabalho preparatório mínima em laboratórios mesmo minimamente equipados 5. Digno de nota, as plaquetas não contêm núcleo e pode, portanto, ser usadas para estudar alterações ao metabolismo independentemente da regulação da transcrição. Aqui nós mostramos queAlém de quantificação relativa de tioésteres de acil-coenzima A (CoA), o sistema de plaquetas isoladas podem ser usadas para analisar o metabolismo de carbono. Especificamente, nós relatamos o uso de marcação metabólica com isótopo estável (não radioativo) marcado [13 C 6] -glucose e [13 C 16] -palmitato para sondar incorporação da [13 C] -label para o importante acetil metabólito CoA através da glicólise ou da oxidação de ácidos gordos. Isto proporciona uma plataforma poderosa, generalizável, e versátil devido ao amplo envolvimento de espécies acil-CoA em vias bioquímicas 13,24 ea rastreabilidade deste sistema para testar outras variáveis, tais como a inibição do complexo I com rotenona 3,33. Além das informações fornecidas no protocolo a seguir, uma extensa descrição dos métodos utilizados para a rotulagem isótopo e para as análises baseadas em LC-MS pode ser encontrado em 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.

Para demonstrar a utilidade desta metodologia reproduzimos a generalização de adaptação metabólica compensatória descrito anteriormente resultantes da exposição a rotenona. Este achado foi identificado previamente em modelos de cultura de células e esta investigação teve como objetivo testar se essa mudança metabólica também ocorre em plaquetas, que são anuclear e não propenso aos mesmos artefatos experimentais como cultura de células. Este trabalho foi realizado com pla...

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

Aqui mostramos plaquetas humanas isoladas podem ser usadas como uma acessível ex vivo modelo para estudar adaptações metabólicas em resposta ao inibidor do complexo I rotenona. Esta abordagem emprega rastreio isotópica e quantificação relativa por espectrometria de massa de cromatografia de líquido e pode ser aplicado a uma variedade de modelos de estudo.

Análise de LC-MS de Plaquetas Humanas como Plataforma para Estudar o Metabolismo Mitocôndrial

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

Education

Research

JoVE Core

JoVE Journal

Statistics

Environment

Unit 1

Measures of Variation

Measures of Relative Standing

Probability Distributions

Estimates

Distributions

Hypothesis Testing

Analysis of Variance

Correlation and Regression

Statistics in Practice

SCIENTISTS IN ACTION

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Técnica de Estudos de Artrópodes e comunidades microbianas dentro Árvore Tecidos

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 ...

Ambiente

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

Expressão Gênica transiente em tabaco usando Assembleia Gibson eo 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

Uso de Chironomidae (Diptera) Pupal Exuviae como um Bioavaliação Protocolo Rápido para massas de água de superfície flutuante

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

Modificação e Aplicação de uma Folha Blower-vac para Campo Amostragem de Artrópodes

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

Térmica varredura Conductometry (TSC) como um método geral para estudar e controlar o comportamento de fase do condutor físico geles

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

Quantificação de proteínas solúveis vegetais e carboidratos digeríveis conteúdo, usando o milho (Zea mays) como um 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

Estudando efeitos neurocomportamentais de poluentes ambientais em larvas de zebrafish

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

O efeito das frações plásticas de resíduos de construção e demolição em propriedades compostas de polímeros de madeira

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

Um sistema gnotobiótico para estudar a montagem do microbioma na Fistofera e na Fermentação Vegetal

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

Projeto de microtrat e preparação de amostras de plantas e solo para 15análises de nitrogênio

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. ...