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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Chemistry

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

Published: May 20th, 2013

DOI:

10.3791/50433

1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania

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.

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.

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 ad....

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1. Sample Extraction from Cells

  1. 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.......

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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).......

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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.......

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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.

....

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

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