Simultaneous, Untargeted Metabolic Profiling of Polar and Nonpolar Metabolites by LC‐Q‐TOF Mass Spectrometry

Jay S. Kirkwood1, Claudia Maier2, Jan F. Stevens1

1 Linus Pauling Institute and Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, 2 Department of Chemistry, Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.39
DOI:  10.1002/0471140856.tx0439s56
Online Posting Date:  May, 2013
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At its most ambitious, untargeted metabolomics aims to characterize and quantify all of the metabolites in a given system. Metabolites are often present at a broad range of concentrations and possess diverse physical properties complicating this task. Performing multiple sample extractions, concentrating sample extracts, and using several separation and detection methods are common strategies to overcome these challenges but require a great amount of resources. This protocol describes the untargeted, metabolic profiling of polar and nonpolar metabolites with a single extraction and using a single analytical platform. Curr. Protoc. Toxicol. 56:4.39.1‐4.39.12. © 2013 by John Wiley & Sons, Inc.

Keywords: untargeted metabolomics; LC‐MS/MS; hypothesis generation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Metabolite Extraction
  • Basic Protocol 2: LC‐MS/MS
  • Basic Protocol 3: Metabolite Identification
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Metabolite Extraction

  • C2C12 mouse muscle cells (plated 24 hr previous in 6‐well plates, 500,000 cells/well)
  • Hank's balanced salt solution (HBSS)
  • Methanol (MeOH; HPLC grade)
  • Ethanol (EtOH; HPLC grade)
  • Plasma aliquots
  • Ice
  • Whole, individual zebrafish at −80°C
  • Tricaine (Tricaine methanesulfonate, CAS# 886‐86‐2)
  • Liquid nitrogen
  • Water (HPLC grade)
  • −80°C freezer
  • Adherent 6‐well plate covers
  • Cell scraper
  • 250‐ml glass beakers
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Glass HPLC vials
  • Mortar
  • Pestle
  • Metal scraper

Basic Protocol 2: LC‐MS/MS

  • Solvent A: Water with 0.1% formic acid
  • Solvent B: Methanol (MeOH) with 0.1% formic acid
  • Calibrant, positive and negative ion solutions (AB SCIEX)
  • Phenyl‐3 HPLC column (Inertsil phenyl‐3, 150 × 4.6 mm, 5 µM)
  • HPLC system (this protocol was carried out using a Nexera system) (Shimadzu)
  • HPLC column oven
  • Q‐TOF mass spectrometer with high‐resolution MS/MS capability (this protocol was carried out using a Triple TOF 5600 equipped with a TurboSpray electrospray ionization source) (AB SCIEX)
  • Glass HPLC vials
  • Calibrant delivery system (CDS; AB SCIEX)
  • MarkerView data processing software (AB SCIEX)

Basic Protocol 3: Metabolite Identification

  • LC‐MS/MS data files
  • Processing computer with internet access
  • PeakView data visualization software (AB SCIEX)
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Literature Cited

Literature Cited
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