Simultaneous, Untargeted Metabolic Profiling of Polar and Nonpolar Metabolites by LC‐Q‐TOF Mass Spectrometry实验方法详情页

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

关键词： simultaneous untargeted metabolic profiling来源：互联网

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

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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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 Materials

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 Materials

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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Figure 4.39.1 (Above) Sample total ion chromatogram (TIC) from rat plasma; (Below) normalized extracted ion chromatograms for several polar and nonpolar metabolites.

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Figure 4.39.2 Example of a PCA‐DA (principal component analysis‐discriminant analysis) scores plot. Legend denotes duration of exposure (skeletal muscle cells) to xanthohumol.

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Figure 4.39.3 Example volcano plot of metabolomics dataset to investigate differences in metabolites for 15 versus 90‐minute treatment times.

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Figure 4.39.4 Metabolite matching by MS and MS/MS.

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Figure 4.39.5 Confirmation of experimental isotope ratio to theoretical isotope ratio.

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Figure 4.39.6 Comparison of experimentally identified metabolite against chemical standard by (A ) LC‐MS and (B ) MS/MS.

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Videos

Literature Cited

Literature Cited
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