Metabolic Profiling and Quantification of Neurotransmitters in Mouse Brain by Gas Chromatography‐Mass Spectrometry

Christian Jäger1, Karsten Hiller1, Manuel Buttini1

1 Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Belvaux
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.15
Online Posting Date:  September, 2016
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Abstract

Metabolites are key mediators of cellular functions, and have emerged as important modulators in a variety of diseases. Recent developments in translational biomedicine have highlighted the importance of not looking at just one disease marker or disease inducing molecule, but at populations thereof to gain a global understanding of cellular function in health and disease. The goal of metabolomics is the systematic identification and quantification of metabolite populations. One of the most pressing issues of our times is the understanding of normal and diseased nervous tissue functions. To ensure high quality data, proper sample processing is crucial. Here, we present a method for the extraction of metabolites from brain tissue, their subsequent preparation for non‐targeted gas chromatography‐mass spectrometry (GC‐MS) measurement, as well as giving some guidelines for processing of raw data. In addition, we present a sensitive screening method for neurotransmitters based on GC‐MS in selected ion monitoring mode. The precise multi‐analyte detection and quantification of amino acid and monoamine neurotransmitters can be used for further studies such as metabolic modeling. Our protocol can be applied to shed light on nervous tissue function in health, as well as neurodegenerative disease mechanisms and the effect of experimental therapeutics at the metabolic level. © 2016 by John Wiley & Sons, Inc.

Keywords: metabolomics; mouse brain; GC‐MS; neurotransmitters; extraction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Metabolic Profiling and Quantification of Neurotransmitters in Mouse Brain with Gas Chromatography‐Mass Spectrometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Metabolic Profiling and Quantification of Neurotransmitters in Mouse Brain with Gas Chromatography‐Mass Spectrometry

  Materials
  • 35 to 80 mg mouse brain tissue
  • 1 M hydrochloric acid (e.g., Sigma‐Aldrich, cat. no. 35328‐1 liter)
  • EF + IS1: Extraction Fluid, containing IS1 (see recipe)
  • Aq. HCl + IS2: Aqueous hydrochloric acid solution, containing IS2 (see recipe)
  • Chloroform, ≥99.9% for HPLC (e.g., CHROMASOLV Plus; Sigma‐Aldrich, cat. no. 650498‐1 liter)
  • Methanol, ≥99.9% for HPLC (e.g., CHROMASOLV; Sigma‐Aldrich, cat. no. 34860‐1L‐R)
  • 200 μM neurotransmitter mix (see recipe)
  • 50 μM neurotransmitter mix (see recipe)
  • Pyridine (Sigma‐Aldrich, cat. no. 270970)
  • Methoxyamine hydrochloride (Sigma‐Aldrich, cat. no. 226904)
  • N‐methyl‐N‐trimethylsilyl‐trifluoroacetamide (MSTFA; Macherey‐Nagel, cat. no. 701270.110)
  • 2‐ml round‐bottom reaction tubes
  • Dry ice or liquid nitrogen
  • Soft tissue homogenizing CK14 kit, 2 ml (Precellys, cat. no. KT03961‐1‐003.2), includes 1.4‐mm ceramic beads in 2‐ml homogenization tubes
  • Precellys24 (Bertin Technologies)
  • Cryolys cooling option for Precellys24 (Bertin Technologies)
  • Thermomixer comfort (Eppendorf)
  • Vial, screw top, amber, silanized (Agilent, cat. no. 5183‐2072)
  • Screw Caps for 2‐ml vials, PTFE/silicone/PTFE (Agilent, cat. no. 5182‐0724)
  • Reaction tube centrifuge (e.g., Eppendorf 5424 R)
  • 2‐ml sample vials with gastight micro insert 250‐5 µl (e.g., CZT, cat. no. 40 11 00 767)
  • Refrigerated rotary vacuum evaporator
  • Agilent J&W DB‐35MS gas chromatography column, 30‐m × 0.25‐mm × 0.25‐μm (L × I.D. × film thickness), or similar
  • Autosampler for automated derivatization (e.g., Gerstel Multi Purpose Sampler)
  • Agilent 7890B GC System – Agilent 5977A inert XL MSD
NOTE: All aqueous solutions used throughout this protocol should be prepared with milliQ or deionized water (18.2 MΩ•cm, <3 ppb TOC). Special attention must be paid to the temperature during sample workup. The whole extraction needs to be performed at 4°C or lower.NOTE: The following procedure was established and optimized for metabolomics analysis of mouse brain. Nevertheless, this method can also be applied for metabolomics analysis of a wide variety of soft tissues, including heart, liver, lung, and muscle. For optimal intensities of chromatographic peaks, the volumes of metabolite extracts transferred into the GC glass vials with micro inserts need to be adjusted.
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Literature Cited

Literature Cited
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