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

We acknowledge support of NIH grants P30ES013508 and 5T32GM008076. We also thank Thermo Scientific for access to SIEVE 2.0 and Drs. Eugene Ciccimaro and Mark Sanders of Thermo Scientific for useful discussions.

1. Sample Extraction from Cells
<ol>
	<li>For a 10 cm plate of cells: collect 1.5 ml of lifted cell suspension in media into a pre-labeled 10 ml glass centrifuge tube. For adherent lines, cells should be lifted with gentle scraping in 1.5 ml of media kept on ice. Optional: If internal standards are used, add an appropriate aliquot at this step. 
	 Comment: Quenching of cellular metabolism is crucial for certain metabolites. For analysis of time-sensitive metabolites, procedures such as cold metha...

<ol>
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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. 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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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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. 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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. 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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. 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Untargeted metabolomics offers a powerful tool for investigating endogenous or xenobiotic biotransformations, or capturing a metabolic profile from a sample of interest. The output of the technique scales with the resolution and sensitivity of the technology used to separate and analyze the sample, the ability to deal with the large datasets generated, and the ability to mine the dataset for useful information (e.g. accurate mass database searching). Recently, this has been facilitated by advances in high resolu...

Due to recent technological advances in the field of HRMS, untargeted, hypothesis-generating metabolomics approaches have become a feasible approach to analysis of complex samples.1 Mass spectrometers capable of 100,000 resolution facilitating routine low part per million (ppm) mass accuracy have become widely available from multiple vendors.2,3 This mass accuracy allows greater specificity and confidence in a preliminary assignment of analyte identity, isotopic pattern recognition, and adduct identification.4 When coupled with an appropriate extraction procedure and high-performance LC or UPLC, complex mixtures can be analyzed with additional specificity derived from retention time data.5 UPLC possesses greater chromatographic efficiency and allows greater sensitivity, resolution and analysis time making a greater coverage of the metabolome possible.6 The resulting large datasets can be integrated into any of multiple differential analysis software and mined for useful patterns or individual analytes of interest.7,8,9,10,11 Putative hits can be initially identified using a combination of peak detection algorithms, accurate mass based chemical formula prediction, fragmentation prediction, and chemical database searching. This approach allows prioritization of targets for time-consuming complete structural identification or for development of more sensitive and more specific stable isotope dilution UPLC/selected or multiple reaction monitoring/MS studies that are the current gold standard methods for quantification.12
The varying nature of biological samples has led to optimization of extraction protocols for urine13, cells14, serum15, or tissue16. This protocol features extractions for cells, serum, and tissue. Where appropriate, comments and additional references have been included for modifications of the procedure to address inclusion of stable isotopes, or for inclusion of especially unstable metabolites.

<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)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

The results presented show selected data from a 6-hr treatment of SH-SY5Y glioblastoma cells with the pesticide and mitochondrial complex I inhibitor rotenone. For brevity, only the organic phase positive mode data is presented. The samples were processed and analyzed as described above (Figure 1, Table 1, Table 2) and loaded onto two differential analysis platforms for label-free quantification, SIEVE and XCMS online. Although a large number of hits (Figure 2, Figure 3)...

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

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (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 ...

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

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

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

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

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

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