Untargeted Metabolomics

Nawaporn Vinayavekhin1, Alan Saghatelian1

1 Harvard University, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 30.1
DOI:  10.1002/0471142727.mb3001s90
Online Posting Date:  April, 2010
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Abstract

Along with genes and proteins, metabolites play important roles in sustaining life. Their functions include “primary” functions in metabolism and energy storage, as well as “secondary” functions in cell‐to‐cell signaling, metal acquisition, and virulence. There remains much to be learned about the in vivo roles of metabolites. Approaches that accelerate measurement of metabolite levels directly from cells and tissues should increase our understanding of the diverse roles of metabolites and potentially lead to discovery of novel metabolites and metabolic pathways. Metabolomics is an important comparative tool to study global metabolite levels in samples under various conditions. In this unit, the steps needed to perform a mass spectrometry (MS)–based untargeted metabolomics experiment using bacterial supernatants are detailed. In contrast to a targeted metabolomics experiment, which measures ions from known metabolites, an untargeted metabolomics experiment registers all ions within a certain mass range, including ions belonging to structurally novel metabolites. The protocols in this unit describe the conditions necessary for analyzing hydrophobic metabolites and provide an example of how to structurally characterize a novel metabolite. Curr. Protoc. Mol. Biol. 90:30.1.1‐30.1.24. © 2010 by John Wiley & Sons, Inc.

Keywords: metabolomics; liquid chromatography–mass spectrometry; secondary metabolites

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Sample Preparation: Isolation of Hydrophobic Metabolites from Bacterial Supernatant
  • Basic Protocol 2: Detecting Hydrophobic Metabolites Using Liquid Chromatography‐Mass Spectrometry (LC‐MS)
  • Basic Protocol 3: Analysis of Untargeted Metabolomics LC‐MS Data
  • Basic Protocol 4: Targeted Data Analysis: Identification and Comparative Analysis of Known Metabolites in Samples
  • Basic Protocol 5: Structural Characterization of Novel Metabolites Using Accurate Mass, Tandem Mass Spectrometry, and Chemical Synthesis
  • Support Protocol 1: Verification of Metabolite: LC‐MS Analysis of Sample, Standard, and Co‐Injection Experiment
  • Support Protocol 2: Tandem Mass Spectrometry Experiment to Detect Fragment Ions of Target Metabolites
  • Support Protocol 3: Addition of Isotopes to Medium to Assist Structure Determination
  • Alternate Protocol 1: Large‐Scale Sample Preparation for Metabolite Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Sample Preparation: Isolation of Hydrophobic Metabolites from Bacterial Supernatant

  Materials
  • Organisms, apparatus, and instruments for growing cultures of interest
  • Chloroform, high purity
  • Methanol, high purity
  • Nitrogen source
  • Refrigerated centrifuge
  • 50‐ml conical tubes
  • Disposable 11‐dram (or 40‐ml) glass vials with caps
  • Disposable 20‐ml glass vials
  • Disposable 1‐dram (or 4‐ml) glass vials with caps
  • 6‐Port Mini‐Vap evaporator/concentrator (Supelco, available from Sigma‐Aldrich; see Fig. A)
NOTE: The protocol is provided for isolating hydrophobic metabolites from a sample. It is imperative to prepare samples from different sets of conditions in parallel and in a consistent manner.

Basic Protocol 2: Detecting Hydrophobic Metabolites Using Liquid Chromatography‐Mass Spectrometry (LC‐MS)

  Materials
  • Metabolite extract sample prepared by protocol 1
  • Water, gas‐chromatography grade
  • Methanol, gas‐chromatography grade
  • 28% (v/v) ammonium hydroxide (NH 4OH; Sigma) in H 2O
  • Isopropanol, gas‐chromatography grade
  • Ammonium formate, 99.995+% (Sigma)
  • Formic acid, ∼98% (Fluka)
  • Autosampler vials with caps (Agilent)
  • 100‐µl or 300‐µl glass inserts for autosampler vials (National Scientific)
  • LC‐MS system (Agilent 6220 LC‐ESI‐TOF instrument; also see unit 10.21)
  • C18 precolumn (3.5 µm, 2 × 20 mm; Upchurch Scientific)
  • C18 (5 µm, 4.6 × 50 mm) column (Gemini column by Phenomenex)
  • C4 precolumn (3.5 µm, 2 × 20 mm; Western Analytical Products, Inc, http://www.westernanalytical.com/)
  • C5 (5 µm, 4.6 × 50 mm) column (Luna column by Phenomenex)
  • Additional reagents and equipment for mass spectrometry (unit 10.21)

Basic Protocol 3: Analysis of Untargeted Metabolomics LC‐MS Data

  Materials
  • Computer
  • Raw data files from LC‐MS experiment ( protocol 2)
  • Software for data conversion (Table 30.1.1)
  • XCMS software (http://masspec.scripps.edu/xcms/xcms.php)

Basic Protocol 4: Targeted Data Analysis: Identification and Comparative Analysis of Known Metabolites in Samples

  Materials
  • Data from LC‐MS experiment ( protocol 2), processed ( protocol 3)

Basic Protocol 5: Structural Characterization of Novel Metabolites Using Accurate Mass, Tandem Mass Spectrometry, and Chemical Synthesis

  Materials
  • Necessary chemicals and solvents for the synthesis
  • Synthetic apparatus
  • Additional reagents and equipment for verification of metabolites ( protocol 6) and MS/MS experiments to detect fragment ions of target metabolites ( protocol 7)

Support Protocol 1: Verification of Metabolite: LC‐MS Analysis of Sample, Standard, and Co‐Injection Experiment

  Materials
  • Synthetic standard for metabolite of interest
  • Metabolite extract sample prepared by protocol 1
  • Disposable 1‐dram (or 4‐ml) glass vials with caps
  • Additional reagents and equipment for LC‐MS ( protocol 2)

Support Protocol 2: Tandem Mass Spectrometry Experiment to Detect Fragment Ions of Target Metabolites

  Materials
  • Synthetic standard for metabolite of interest
  • Chloroform, high purity
  • Metabolite extract sample prepared by protocol 1
  • Disposable 1‐dram (or 4‐ml) glass vials with caps
  • LC‐MS system that can perform MS/MS experiment (e.g., Agilent 6520 LC‐ESI‐QTOF instrument)
  • Additional reagents and equipment for LC‐MS ( protocol 2)

Support Protocol 3: Addition of Isotopes to Medium to Assist Structure Determination

  • 13C‐labeled or deuterated intermediate in the biosynthetic pathway of metabolite of interest

Alternate Protocol 1: Large‐Scale Sample Preparation for Metabolite Analysis

  • 250‐ml or 500‐ml centrifuge bottles
  • 1‐liter glass bottles
  • 50‐ml glass graduated cylinders
  • 100‐ml glass graduated cylinders
  • 500‐ml separatory funnel
  • 250‐ml round‐bottom flasks
  • Rotary evaporator
NOTE: The protocol provided is for the isolation of hydrophobic metabolites from a sample. It is imperative to prepare samples in parallel and in a consistent manner to provide the most accurate analysis possible.
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Figures

  •   FigureFigure 30.1.1 Overall workflow for an untargeted metabolomics analysis.
  •   FigureFigure 30.1.2 Simplified scheme for sample preparation presented in .
  •   FigureFigure 30.1.3 Nitrogen gas blowers (6‐Port Mini‐Vap evaporator/concentrator; Sigma‐Aldrich). For ease of use, evaporators obtained from a commercial source (A) can be assembled onto a support jack before connecting to the nitrogen source (B). The apparatus allows rapid evaporation of organic solvent (chloroform) from multiple samples at once.
  •   FigureFigure 30.1.4 Examples of extracted ion chromatograms showing false positive (left) and real peak (right).
  •   FigureFigure 30.1.5 Examples of calculation for steps 4 to 5 in .
  •   FigureFigure 30.1.6 An isotope pattern of 2′‐(2‐hydroxyphenyl)‐4′‐thiazolyl‐2,4‐thiazolinyl‐4‐carboxylic acid (HPTzTn‐COOH) detected in the culture of Pseudomonas aeruginosa PA14 WT strain grown in M9 minimal media in the presence of DL‐cysteine‐3,3‐ d2. The detected mass‐to‐charge ratios ( m/ z) correspond to the incorporation of DL‐cysteine‐3,3‐ d2 into the first ring, second ring, or both rings of the metabolite, resulting in the addition of one, two, or three deuteriums, respectively.

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