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

Vi anerkender støtte til NIH tilskud P30ES013508 og 5T32GM008076. Vi takker også Thermo Scientific for adgang til SIEVE 2.0 og Drs. Eugene Ciccimaro og Mark Sanders Thermo Scientific for nyttige drøftelser.

1.. Sample Udtræk fra Cells <ol><li> For en 10 cm plade af celler: indsamle 1,5 ml løftet cellesuspension i medier i en præ-mærket 10 ml glas centrifugerør. For vedhængende linjer bør celler løftes med blide skrabning i 1,5 ml af medier holdes på is Valgfrit:. Hvis de interne standarder anvendes, tilsættes en passende portion på dette trin. Kommentar: Bratkøling af cellulær metabolisme er afgørende for visse metabolitter. Til analyse af tidsfølsomme metabolitter, bør procedurer...

<ol>
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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. 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Målrettede metabolomics tilbyder et kraftfuldt værktøj til at undersøge endogene eller miljøfremmede biotransformationer, eller indfange en metabolisk profil fra en prøve af interesse. Udgangen af teknikken skalaer med opløsning og følsomhed af den teknologi, der anvendes til at adskille og analysere prøverne, evnen til at håndtere de store genererede datasæt, og evnen til at udvinde datasættet for nyttige oplysninger (f.eks nøjagtige masse database søgning). For nylig har dette blevet fremmet af ...

Grund af de seneste teknologiske fremskridt inden for HRMS har målrettede, hypotese-genererende metabolomics tilgange blevet en realistisk tilgang til analyse af komplekse prøver. 1 massespektrometre stand 100.000 resolution lette rutinemæssig lave del per million (ppm) masse nøjagtighed blevet bredt tilgængelige fra flere leverandører. 2,3 Denne masse nøjagtighed tillader større specificitet og tillid i en indledende tildeling af analyt identitet, isotop mønstergenkendelse og adduct identifikation. 4. Når kombineret med en passende ekstraktionsprocedure og højtydende LC eller UPLC, komplekse blandinger kan analyseres med ekstra specificitet stammer fra fastholdelse tid data. 5. UPLC besidder større kromatografisk effektivitet og giver større følsomhed, opløsning og analyse tid på at gøre en større dækning af metabolomet muligt. 6. De resulterende store datasæt kan integreres i ethvertaf multiple differential analyse software og udvindes til nyttige mønstre eller individuelle analytter af interesse. 7,8,9,10,11 Formodede hits kan i første omgang identificeres ved hjælp af en kombination af peak detekteringsalgoritmer, præcis masse baseret kemiske formel forudsigelse, fragmentering forudsigelse og kemisk database søgning. Denne fremgangsmåde giver mulighed for prioritering af mål for tidskrævende fuldstændig strukturel identifikation eller af udvikling af mere følsomme og mere specifikke stabil isotopfortynding UPLC / udvalgte eller multipel reaktion overvågning / MS undersøgelser, som de nuværende guld standard metoder til kvantificering. 12. De forskellige former for biologiske prøver har ført til optimering af udvinding protokoller for urin 13 celler 14, serum 15 eller væv 16. Denne protokol funktioner ekstraktioner for celler, serum og væv. I givet fald har kommentarer og yderligere henvisninger er medtaget for ændrintioner af proceduren for at løse inddragelse af stabile isotoper, eller om optagelse af særligt ustabile metabolitter.

<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)

Her præsenterer vi en arbejdsproces til at analysere de metaboliske profiler til biologiske prøver af interesse, herunder, celler, serum eller væv. Prøven først adskilles i polære og ikke-polære fraktioner ved en væske-væske fase ekstraktion, og delvist oprenset for at lette downstream analyse. Både vandige (polære metabolitter) og organisk (ikke-polære metabolitter) faser af indledende ekstraktion behandles for at undersøge en bred vifte af metabolitter. Metabolitter adskilt af forskellige flydende kromatografiske metoder baseret på deres partition egenskaber. I denne metode, præsenterer vi Microflow ultra-performance (UP) LC-metoder, men protokollen er skalerbar til højere strømme og lavere tryk. Introduktion i massespektrometret kan være gennem enten generelle eller sammensatte optimerede kilde betingelser. Påvisning af en bred vifte af ioner udføres i fuld scanning i både positiv og negativ modus over et bredt m / z rækkevidde ved hjælp af høj opløsning på en nylig calibrated instrument. Label-fri differentieret analyse udføres på bioinformatik platforme. Anvendelser af denne fremgangsmåde omfatter metaboliseringsvej screening, biomarkør opdagelse og udvikling af lægemidler.

De præsenterede resultater viser udvalgte data fra en 6 timers behandling af SH-SY5Y glioblastomceller med pesticidet og mitokondrie kompleks jeg inhibitor rotenon. For kortheds skyld, er det kun den organiske fase positive tilstand data præsenteres. Prøverne blev behandlet og analyseret som beskrevet ovenfor (figur 1, tabel 1, tabel 2) og sat på to differential analyse platforme til etiket-fri kvantificering, Si og XCMS online. Selv om et stort antal hits (figur 2, figur 3) er iden...

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

Målrettede metabolomics giver en hypotese genererer øjebliksbillede af en metabolisk profil. Denne protokol vil demonstrere ekstraktion og analyse af metabolitter fra celler, serum eller væv. En række metabolitter undersøges ved hjælp af væske-væske fase ekstraktion, Microflow ultraperformance væskekromatografi / høj opløsning massespektrometri (UPLC-HRMS) koblet til differential analyse software.

Målrettede Metabolomics fra biologiske kilder Brug Ultraperformance væskekromatografi-højopløsningsmassespektrometri (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 Ikke-målrettet metabolomisk profilering af Serum fra Ultra Performance Liquid Chromatography-massespektrometri (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

Analyserer Protein Dynamics hjælp af brint Exchange massespektrometri

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

Hurtig High-throughput Arter Identifikation af Botanisk Materiale Brug Direct Analyse i Real Time High Resolution massespektrometri

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

En HS-MRM analyse til kvantificering af værts-celle proteiner i Protein Biopharmaceuticals af væskekromatografi Ion mobilitet QTOF massespektrometri

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 ånde analyse ved hjælp af sekundære Nanoelectrospray ionisering koblet til høj opløsning massespektrometri

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

Syntese og massespektrometri analyse af 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 kapillær elektroforese massespektrometri for encellede Metabolomics i Live Frog (Xenopus laevis) embryoner

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

Storstilet Top-down Proteomics brug af kapillar Zone elektroforese Tandem massespektrometri

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

Høj overførselshastighed og omfattende stof overvågning ved hjælp af Multisegment injektion-kapillær elektroforese massespektrometri

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