Name: untargeted metabolomics from biological sources using ultraperformance liquid chromatography-high resolution mass spectrometry (uplc-hrms)
Uploaded: 2013-05-20T14:00:00
Description: Watch this Scientific Journal Video about Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS at JoVE.com

Wij erkennen de steun van NIH beurzen P30ES013508 en 5T32GM008076. We danken ook Thermo Scientific voor de toegang tot ZEEF 2.0 en Drs. Eugene Ciccimaro en Mark Sanders van Thermo Scientific voor nuttige discussies.

1. Staalextractie uit cellen <ol><li> Voor een 10 cm plaat van cellen: verzamel 1,5 ml celsuspensie opgeheven in media in een pre-gelabelde 10 ml glazen centrifugebuis. Voor aanhangend lijnen, moeten cellen worden opgeheven met zachte schrapen in 1,5 ml van media op ijs bewaard Optioneel:. Als interne standaarden worden gebruikt, voeg een passende hoeveelheid bij deze stap. Commentaar: Het doven van de cellulaire stofwisseling is cruciaal voor bepaalde metabolieten. Voor de analyse van tijdgev...

<ol>
	<li>Pluskal, T., Nakamura, T., Villar-Briones, A., Yanagida, M. <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+of+the+fission+yeast+S.+pombe:+quantification+of+compounds+under+different+temperatures+and+genetic+perturbation.">Metabolic profiling of the fission yeast S. pombe: quantification of compounds under different temperatures and genetic perturbation.</a> Mol. Biosyst. 6 (1), 182-198 (2010).</li><li>Makarov, A., Denisov, E., 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=Performance+Evaluation+of+a+Hybrid+Linear+Ion+Trap/Orbitrap+Mass+Spectrometer.">Performance Evaluation of a Hybrid Linear Ion Trap/Orbitrap Mass Spectrometer.</a> Analytical Chemistry. 78 (7), 2113-2120 (2006).</li><li>Timischl, B., Dettmer, K., Kaspar, H., Thieme, M., Oefner, P. J. <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+quantitative,+validated+capillary+electrophoresis-time+of+flight-mass+spectrometry+method+with+integrated+high-confidence+analyte+identification+for+metabolomics.">Development of a quantitative, validated capillary electrophoresis-time of flight-mass spectrometry method with integrated high-confidence analyte identification for metabolomics.</a> Electrophoresis. 29 (10), 2203-2214 (2008).</li><li>Katajamaa, M., Oresic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+processing+for+mass+spectrometry-based+metabolomics.">Data processing for mass spectrometry-based metabolomics.</a> J. Chromatogr. A. 1158 (1-2), 318-328 (2007).</li><li>Katajamaa, M., Oresic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+methods+for+differential+analysis+of+LC/MS+profile+data.">Processing methods for differential analysis of LC/MS profile data.</a> BMC Bioinformatics. 6, 179 (2005).</li><li>Wilson, I. D., Nicholson, 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=High+resolution+ultra+performance+liquid+chromatography+coupled+to+q-TOF+mass+spectrometry+as+a+tool+for+differential+metabolic+pathway+profiling+in+functional+genomic+studies.">High resolution ultra performance liquid chromatography coupled to q-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies.</a> Journal of Proteome Research. 4 (2), 591-598 (2005).</li><li>Benton, H. P., Wong, D. M., Trauger, S. A., Siuzdak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XCMS2:+processing+tandem+mass+spectrometry+data+for+metabolite+identification+and+structural+characterization.">XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization.</a> Anal. Chem. 80 (16), 6382-6389 (2008).</li><li>Katajamaa, M., Miettinen, J., Oresic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MZmine:+toolbox+for+processing+and+visualization+of+mass+spectrometry+based+molecular+profile+data.">MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data.</a> Bioinformatics. 22 (5), 634-636 (2006).</li><li>Pluskal, T., Castillo, S., Villar-Briones, A., Oresic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MZmine+2:+Modular+framework+for+processing,+visualizing,+and+analyzing+mass+spectrometry-based+molecular+profile+data.">MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data.</a> BMC Bioinformatics. 11 (1), 395 (2010).</li><li>Smith, C. A., Want, E. J., O'Maille, G., Abagyan, R., Siuzdak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XCMS:+Processing+Mass+Spectrometry+Data+for+Metabolite+Profiling+Using+Nonlinear+Peak+Alignment,+Matching,+and+Identification.">XCMS: Processing Mass Spectrometry Data for Metabolite Profiling Using Nonlinear Peak Alignment, Matching, and Identification.</a> Analytical Chemistry. 78 (3), 779-787 (2006).</li><li>Tautenhahn, R., Patti, G. J., Rinehart, D., Siuzdak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XCMS+Online:+A+Web-Based+Platform+to+Process+Untargeted+Metabolomic+Data.">XCMS Online: A Web-Based Platform to Process Untargeted Metabolomic Data.</a> Analytical Chemistry. 84 (11), 5035-5039 (2012).</li><li>Gelhaus, S. L., Mesaros, A. 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=Cellular+Lipid+Extraction+for+Targeted+Stable+Isotope+Dilution+Liquid+Chromatography-Mass+Spectrometry+Analysis.">Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis.</a> J. Vis. Exp. (57), e3399 (2011).</li><li>Want, E. J., Wilson, I. D., 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=Global+metabolic+profiling+procedures+for+urine+using+UPLCG&#199;&#244;MS.">Global metabolic profiling procedures for urine using UPLCG&#199;&#244;MS.</a> Nature Protocols. 5 (6), 1005-1018 (2010).</li><li>Sellick, C. A., Hansen, R., Stephens, G. M., Goodacre, R., Dickson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolite+extraction+from+suspension-cultured+mammalian+cells+for+global+metabolite+profiling.">Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling.</a> Nature Protocols. 6 (8), 1241-1249 (2011).</li><li>Dunn, W. B., Broadhurst, D., 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=Procedures+for+large-scale+metabolic+profiling+of+serum+and+plasma+using+gas+chromatography+and+liquid+chromatography+coupled+to+mass+spectrometry.">Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry.</a> Nature protocols. 6 (7), 1060-1083 (2011).</li><li>Masson, P., Alves, A. C., Ebbels, T. M. D., Nicholson, J. K., Want, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+evaluation+of+metabolite+extraction+protocols+for+untargeted+metabolic+profiling+of+liver+samples+by+UPLC-MS.">Optimization and evaluation of metabolite extraction protocols for untargeted metabolic profiling of liver samples by UPLC-MS.</a> Analytical Chemistry. 82 (18), 7779-7786 (2010).</li><li>Shaham, O., Slate, N. 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=A+plasma+signature+of+human+mitochondrial+disease+revealed+through+metabolic+profiling+of+spent+media+from+cultured+muscle+cells.">A plasma signature of human mitochondrial disease revealed through metabolic profiling of spent media from cultured muscle cells.</a> Proceedings of the National Academy of Sciences. 107 (4), 1571-1575 (2010).</li><li>Cequier-Sa&#252;nchez, E., Rodri&#252;guez, C., Ravelo, A. G., Za&#252;rate, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dichloromethane+as+a+Solvent+for+Lipid+Extraction+and+Assessment+of+Lipid+Classes+and+Fatty+Acids+from+Samples+of+Different+Natures.">Dichloromethane as a Solvent for Lipid Extraction and Assessment of Lipid Classes and Fatty Acids from Samples of Different Natures.</a> Journal of Agricultural and Food Chemistry. 56 (12), 4297-4303 (2008).</li><li>Keller, A., Eng, J., Zhang, N., Li, X. J., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+uniform+proteomics+MS/MS+analysis+platform+utilizing+open+XML+file+formats.">A uniform proteomics MS/MS analysis platform utilizing open XML file formats.</a> Mol. Syst. Biol. 1, (2005).</li><li>Cleveland, W. S., Devlin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+weighted+regression+-+an+approach+to+regression-analysis+by+local+fitting.">Locally weighted regression - an approach to regression-analysis by local fitting.</a> J. Am. Stat. Assoc. 83 (403), 596-610 (1988).</li><li>Lange, E., Tautenhahn, R., Neumann, S., Gropl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+assessment+of+alignment+procedures+for+LC-MS+proteomics+and+metabolomics+measurements.">Critical assessment of alignment procedures for LC-MS proteomics and metabolomics measurements.</a> BMC Bioinformatics. 9 (1), 375 (2008).</li><li>Tautenhahn, R., Bottcher, C., Neumann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+feature+detection+for+high+resolution+LC/MS.">Highly sensitive feature detection for high resolution LC/MS.</a> BMC Bioinformatics. 9 (1), 504 (2008).</li><li>Kuhl, C., Tautenhahn, R., Bottcher, C., Larson, T. R., Neumann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAMERA:+An+Integrated+Strategy+for+Compound+Spectra+Extraction+and+Annotation+of+Liquid+Chromatography/Mass+Spectrometry+Data+Sets.">CAMERA: An Integrated Strategy for Compound Spectra Extraction and Annotation of Liquid Chromatography/Mass Spectrometry Data Sets.</a> Analytical Chemistry. 84 (1), 283-289 (2012).</li><li>Kanehisa, M., Goto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KEGG:+kyoto+encyclopedia+of+genes+and+genomes.">KEGG: kyoto encyclopedia of genes and genomes.</a> Nucleic Acids Res. 28 (1), 27-30 (2000).</li><li>Smith, C. A., O'Maille, 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=METLIN:+a+metabolite+mass+spectral+database.">METLIN: a metabolite mass spectral database.</a> Ther. Drug Monit. 27 (6), 747-751 (2005).</li><li>Wang, Y., Xiao, J., Suzek, T. O., Zhang, J., Wang, J., Bryant, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PubChem:+a+public+information+system+for+analyzing+bioactivities+of+small+molecules.">PubChem: a public information system for analyzing bioactivities of small molecules.</a> Nucleic Acids Res. 37 Web Server, W623-W633 (2009).</li><li>Wishart, D. S., Knox, 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=HMDB:+a+knowledgebase+for+the+human+metabolome.">HMDB: a knowledgebase for the human metabolome.</a> Nucleic Acids Res. 37 Database, D603-D610 (2009).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Canadian Journal of Biochemistry and Physiology. 37 (8), 911-917 (1959).</li><li>Folch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipids+from+animal+tissues.">A simple method for the isolation and purification of total lipids from animal tissues.</a> J. Biol. Chem. 226, 497-509 (1957).</li><li>Avery, 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=Quantitative+characterization+of+differential+ion+suppression+on+liquid+chromatography/atmospheric+pressure+ionization+mass+spectrometric+bioanalytical+methods.">Quantitative characterization of differential ion suppression on liquid chromatography/atmospheric pressure ionization mass spectrometric bioanalytical methods.</a> Rapid Communications in Mass Spectrometry. 17 (3), 197-201 (2003).</li></ol>

Ongerichte metabolomics biedt een krachtig hulpmiddel voor het onderzoeken van endogene of lichaamsvreemde biotransformaties, of het vastleggen van een metabool profiel van een steekproef van belang. De uitgang van de techniek schalen met de resolutie en de gevoeligheid van de gebruikte te scheiden en analyseren van het monster, het vermogen om te gaan met de grote datasets gegenereerd technologie, en de mogelijkheid om de dataset voor nuttige informatie (bv. accurate massa zoeken in databanken) delven. Onlangs...

Als gevolg van recente technologische ontwikkelingen op het gebied van HRMS, hebben ongerichte, hypothese-genererende metabolomics aanpak een haalbare aanpak worden voor de analyse van complexe monsters. 1 massaspectrometers staat 100000 resolutie vergemakkelijken routine lage deel per miljoen (ppm) nauwkeurigheid van massa&#39;s zijn op grote schaal te worden beschikbaar zijn van meerdere leveranciers. 2,3 Dit nauwkeurigheid van massa mogelijk maakt grotere specificiteit en het vertrouwen in een voorlopige toewijzing van de identiteit analyt, isotopische patroonherkenning, en adduct identificatie. 4 In combinatie met een geschikte extractiemethode en high-performance LC of UPLC, complexe mengsels kunnen worden geanalyseerd met extra specificiteit afgeleid van retentietijdgegevens. 5 UPLC bezit chromatografische grotere efficiëntie en maakt een grotere gevoeligheid, resolutie en analysetijd die een grotere dekking van de mogelijke metabolome. 6 De resulterende grote datasets kan worden geïntegreerd in elkevan meerdere differentiële analyse software en ontgonnen bruikbare patronen of individueel te bepalen analyten. 7,8,9,10,11 Vermeende treffers kan in eerste instantie worden geïdentificeerd met behulp van een combinatie van piek-detectie-algoritmen, accurate massa gebaseerde chemische formule voorspelling, fragmentatie voorspelling, en chemische zoeken in databanken. Deze aanpak maakt prioritering van doelstellingen voor tijdrovende volledige structurele identificatie of voor de ontwikkeling van gevoeliger en specifieker stabiele isotoop verdunning UPLC / geselecteerde of meerdere reactie bewaking / MS studies dat de huidige gouden standaard methoden voor het kwantificeren zijn. 12 De wisselende aard van de biologische monsters heeft geleid tot optimalisatie van extractie protocollen voor urine 13 cellen 14, 15 serum of weefsel 16. Dit protocoleigenschappen extracties voor cellen, serum en weefsel. In voorkomend geval, zijn commentaar en extra verwijzingen opgenomen voor aanpasgen van de procedure opname van stabiele isotopen pakken, of voor opname van bijzonder onstabiel metabolieten.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Mediatech</td>
			<td>21-031-CM</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Water (H2O)</td>
			<td>Fisher Scientific</td>
			<td>W7-4</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Acetonitrile (CH3CN)</td>
			<td>Fisher Scientific</td>
			<td>A996-4</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Methanol (CH3OH)</td>
			<td>Fisher Scientific</td>
			<td>A454-4</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A464-4</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Chloroform (CH3Cl)</td>
			<td>Sigma-Aldrich</td>
			<td>366927</td>
			<td>Hazard</td>
		</tr>
		<tr>
			<td>Dichloromethane (CH2Cl2)</td>
			<td>Acros Organics</td>
			<td>61030-1000</td>
			<td>To replace chloroform</td>
		</tr>
		<tr>
			<td>Diethyl Ether</td>
			<td>Sigma-Aldrich</td>
			<td>346136</td>
			<td>To replace chloroform</td>
		</tr>
		<tr>
			<td>Formic Acid (FA)</td>
			<td>Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>NH4OH</td>
			<td>Fisher Scientific</td>
			<td>A470-250</td>
			<td>(optima)</td>
		</tr>
		<tr>
			<td>Ammonium formate (HCOONH4)</td>
			<td>Sigma-Aldrich</td>
			<td>78314</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MicroSpin C18 Columns</td>
			<td>Nest Group Inc</td>
			<td>SS18V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pasteur Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml Glass Centrifuge Tubes</td>
			<td>Kimble Chase</td>
			<td>73785-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml Plastic Centrifuge Tubes</td>
			<td>CellTreat</td>
			<td>CLS-4301-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LC Vials (glass)</td>
			<td>Waters</td>
			<td>60000751CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LC Inserts (glass)</td>
			<td>Waters</td>
			<td>WAT094171</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LC Vials (plastic)</td>
			<td>Waters</td>
			<td>186002640</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.22 &mu;m Filters</td>
			<td>Corning</td>
			<td>8169</td>
			<td>nylon</td>
		</tr>
		<tr>
			<td>2 ml Eppendorf Tubes</td>
			<td>BioExpress</td>
			<td>C-3229-1</td>
			<td>Low Retention</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>High Resolution Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>LTQ XL-Orbitrap</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC/UPLC</td>
			<td>Waters</td>
			<td>nanoACQUITY UPLC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Source</td>
			<td>Michrom</td>
			<td>Thermo Advance Source</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Differential Analysis Software</td>
			<td>Thermo Scientific</td>
			<td>SIEVE 2.0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>nanoACQUITY C18 BEH130</td>
			<td>Waters</td>
			<td>186003546</td>
			<td>1.7 &mu;m particle size, 150 mm x 100 &mu;m</td>
		</tr>
		<tr>
			<td>Acentis Express C8</td>
			<td>Sigma-Aldrich</td>
			<td>54262</td>
			<td>2.7 &mu;m particle size, 15 cm x 200 &mu;m</td>
		</tr>
	</tbody>
</table>

untargeted metabolomics from biological sources using ultraperformance liquid chromatography-high resolution mass spectrometry (uplc-hrms)

Hier presenteren we een workflow om de metabolische profielen te analyseren voor biologische monsters van belang, met inbegrip, cellen, serum of weefsel. Het monster wordt eerst gescheiden in polaire en niet-polaire fracties met een vloeistof-vloeistof fase-extractie, en gedeeltelijk gezuiverd downstream analyse te vergemakkelijken. Beide waterige (polaire metabolieten) en organische (niet-polaire metabolieten) fasen van de aanvankelijke extractie worden verwerkt tot een groot aantal metabolieten onderzoeken. Metabolieten worden gescheiden door verschillende vloeistofchromatografie methoden gebaseerd op hun partitie eigenschappen. In deze methode presenteren we microflow hoogperformante (UP) LC methoden, maar het protocol is schaalbaar tot hogere stromen en lagere drukken. Inleiding in de massaspectrometer kan via algemene of samengestelde geoptimaliseerde bron voorwaarden. Detectie van een breed scala van ionen wordt in volledige scan mode uitgevoerd in zowel positieve als negatieve wijze over een breed m / z assortiment met een hoge resolutie op een recent calibrated instrument. Label-free differentiële analyse wordt uitgevoerd op bioinformatica platforms. Toepassingen van deze aanpak zijn metabole route screening, de ontdekking van biomarkers en de ontwikkeling van geneesmiddelen.

De gepresenteerde resultaten tonen geselecteerde gegevens uit een 6-uur behandeling van SH-SY5Y glioblastoma cellen met het bestrijdingsmiddel en mitochondriaal complex I remmer rotenone. Kortheidshalve wordt alleen de organische fase positieve modus gepresenteerde gegevens. De monsters werden verwerkt en geanalyseerd zoals hierboven beschreven (Figuur 1, Tabel 1, Tabel 2) en aangebracht op twee differentiële analyseplatforms voor label-free kwantificering ZEEF en XCMS online. Hoewel een groot aantal h...

Watch this Scientific Journal Video about Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS at JoVE.com

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Ongerichte metabolomics biedt een hypothese genererende momentopname van een metabool profiel. Dit protocol zal de extractie en analyse van metabolieten uit cellen, serum of weefsel tonen. Een reeks van metabolieten worden onderzocht met behulp van vloeistof-vloeistof fase-extractie, microflow UltraPerformance vloeistofchromatografie / hoge-resolutie massaspectrometrie (UPLC-HRMS) gekoppeld aan differentiële analyse software.

Ongerichte Metabolomics uit biologische grondstoffen gebruiken UltraPerformance Liquid Chromatography-hoge resolutie massaspectrometrie (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Research

JoVE Journal

Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Large Scale Niet-gerichte metaboloom profilering van Serum door Ultra Performance Liquid Chromatography-massaspectrometrie (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

Het analyseren van Protein Dynamics gebruik van waterstof Exchange Massaspectrometrie

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

Rapid High-throughput Species Identificatie van Botanical Material gebruik van Direct Analysis in Real Time met hoge resolutie massaspectrometrie

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

Een HS-MRM Assay voor de kwantificering van de gastheercel eiwitten in Protein biofarmaceutica door vloeistofchromatografie Ion mobiliteit QTOF massaspectrometrie

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Real-time adem analyse met behulp van secundaire Nanoelectrospray ionisatie gekoppeld aan massaspectrometrie voor hoge resolutie

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Synthese en analyse van de Spectrometrie van de massa van Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

Microprobe capillaire elektroforese massaspectrometrie voor eencellige metabolomica in Live kikker (Xenopus laevis) embryo 's

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Grootschalige topdown Proteomics met behulp van capillaire Zone elektroforese Tandem massaspectrometrie

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

High-throughput en uitgebreide Drug Surveillance met behulp van spectrometrie van de massa van het Multisegment injectie-capillaire elektroforese

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...