Metabolomics by Gas Chromatography–Mass Spectrometry: Combined Targeted and Untargeted Profiling

Oliver Fiehn1

1 King Abdulaziz University, Biochemistry Department, Jeddah, Saudi Arabia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 30.4
DOI:  10.1002/0471142727.mb3004s114
Online Posting Date:  April, 2016
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Abstract

Gas chromatography–mass spectrometry (GC‐MS)–based metabolomics is ideal for identifying and quantitating small‐molecule metabolites (<650 Da), including small acids, alcohols, hydroxyl acids, amino acids, sugars, fatty acids, sterols, catecholamines, drugs, and toxins, often using chemical derivatization to make these compounds sufficiently volatile for gas chromatography. This unit shows how GC‐MS‐based metabolomics allows integration of targeted assays for absolute quantification of specific metabolites with untargeted metabolomics to discover novel compounds. Complemented by database annotations using large spectral libraries and validated standard operating procedures, GC‐MS can identify and semiquantify over 200 compounds from human body fluids (e.g., plasma, urine, or stool) per study. Deconvolution software enables detection of more than 300 additional unidentified signals that can be annotated through accurate mass instruments with appropriate data processing workflows, similar to untargeted profiling using liquid chromatography–mass spectrometry. GC‐MS is a mature technology that uses not only classic detectors (quadrupole) but also target mass spectrometers (triple quadrupole) and accurate mass instruments (quadrupole–time of flight). This unit covers sample preparation from mammalian samples, data acquisition, quality control, and data processing. © 2016 by John Wiley & Sons, Inc.

Keywords: GC‐MS; mass spectrometry; compound identification; structure elucidation; pathway mapping; precision medicine; multivariate statistics

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Mammalian Samples for GC‐MS Metabolome Analysis
  • Support Protocol 1: Preparation of External and Internal Reference Standards for Quality Control
  • Basic Protocol 2: GC‐MS Data Acquisition for Metabolome Analysis
  • Basic Protocol 3: GC‐MS Raw Data Quality Control for Metabolome Analysis
  • Basic Protocol 4: GC‐MS Data Processing For Metabolome Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Mammalian Samples for GC‐MS Metabolome Analysis

  Materials
  • Acetonitrile, LCMS grade
  • Isopropanol, LCMS grade
  • Ultrapure water, <18 mΩ residual conductivity
  • Internal standards
  • Nitrogen gas line with glass pipet tip
  • Biological sample: blood plasma or serum, urine, cell culture, or homogenized tissue
  • Reference QC standards (e.g., NIST standard blood plasma SRM1950)
  • External reference standards QC mix (see protocol 2Support Protocol)
  • Methoxyamine hydrochloride (MeOX)
  • Pyridine
  • N‐Methyl‐N‐(trimethylsilyl)trifluoroacetamide (MSTFA)
  • FAME internal marker solution (see protocol 2Support Protocol)
  • Solvent cooling bath, –20°C
  • 1.5‐ml polypropylene microcentrifuge tubes, uncolored (e.g., Eppendorf PCR tubes with hinged safe‐locks)
  • Crushed ice bath kept <0°C by with NaCl
  • Vortex
  • Microcentrifuge (e.g., Labconco Centrivap)
  • Nitrogen evaporator
  • Degassing device
  • SpeedVac evaporator
  • Sonicator
  • Shaker for 1.5‐ml tubes
  • 2‐ml glass autosampler crimp vials, conical or equipped with micro‐inserts, with Teflonized seals
  • Autosampler crimper and decapper
  • Orbital mixing chilling/heating plate
NOTE: Colored sample tubes should not be used, as they may leak contaminant chemicals into the mixture. The microcentrifuge should have a phenol‐free lid seal and built‐in vacuum delay to prevent bumping by allowing the rotor to achieve speed before applying vacuum.NOTE: For urine samples, normalize the extraction volume for each sample to clinical creatinine levels (a measure of glomerular filtration rate) or osmolality levels (a measure of the total concentration of all solutes, including salts and metabolites). Concentrations of metabolites in urine vary drastically, e.g., influenced by the total volume of liquids a subject has consumed in the hours before urine collection. In order to have a similar number of metabolites detected in urine, and to avoid saturating the detector, the volume of urine used for metabolite extractions should be controlled for the total concentration of metabolites prior to data acquisition. Creatinine or osmolality are regarded as good surrogate measures for this total urinary metabolite concentration.

Support Protocol 1: Preparation of External and Internal Reference Standards for Quality Control

  Materials
  • Ultrapure water, <18 mΩ residual conductivity
  • Methanol, LCMS grade
  • Isopropanol, LCMS grade
  • Nitrogen gas line with glass pipet tip
  • External reference standards (Table 30.4.1)
  • Fatty acid methyl ester (FAME) internal reference standards (Table 30.4.2)
  • Chloroform
  • 25‐ and 250‐ml volumetric flasks with glass stoppers and stir bars
  • Precision balance (accuracy ±0.1 mg)
  • 1.5‐ml glass vials
  • Amber bottle, ≥25 ml
Table 0.4.1   MaterialsCompounds in External Reference QC MixPreparation and Use of FAME Mixture for Internal Standards

Compound a Retention time (sec) Solvent Weight (mg)
Pyruvate 6.74 Water 10.00
Alanine 7.53 Water 10.00
Valine 9.16 Water 10.00
Serine 9.74 Water 10.00
Nicotinic acid 10.258 Water 10.00
Succinic acid 10.52 Water 10.00
Methionine 11.82 Water 20.00 b
Aspartic acid 12 Solution A 20.00 b
4‐Hydroxyproline 12.62 Water 10.00
Salicylic acid 13.089 Water 10.00
Glutamic acid 13.37 Solution A 10.00
Creatinine 13.66 Water 10.00
α‐Ketoglutaric acid 13.86 Water 10.00
N‐Acetylaspartic acid 14.8 Water 10.00
Asparagine 14.97 Water 10.00
Putrescine 15.77 Water 10.00
Shikimic acid 16.493 Water 10.00
Citric acid 16.63 Water 10.00
Lysine 16.975 Water 10.00
D‐(+)‐Glucose 17.47 Water 10.00
Glucose‐6‐phosphate 21.381 Water 10.00
Arachidic acid 22.364 Chloroform 10.00
Serotonin 22.51 Methanol 10.00
Adenosine 23.862 Solution A 10.00
Sucrose 23.95 Water 10.00
Chlorogenic acid 26.39 Methanol 10.00
α‐Tocopherol 27.397 Chloroform 10.00
Cholesterol 27.528 Chloroform 10.00
Compound Fiehn retention index value Kovats retention index value Amount (mg) Concentration (mg/ml)
Methyl caprylate/octanoate (C08) 262320 1083 20 0.8
Methyl pelargonate/nonanoate (C09) 323120 1183 20 0.8
Methyl caprate/decanoate (C10) 381020 1282 20 0.8
Methyl laurate/dodecanoate (C12) 487220 1481 20 0.8
Methyl myristate/tetradecanoate (C14) 582620 1680 20 0.8
Methyl palmitate/hexadecanoate (C16) 668720 1878 20 0.8
Methyl stearate/octadecanoate (C18) 747420 2077 10 0.4
Methyl arachidate/icosanoate (C20) 819620 2276 10 0.4
Methyl behenate/docosanoate (C22) 886620 2475 10 0.4
Methyl tetracosanoate (C24) 948820 2674 10 0.4
Methyl hexacosanoate (C26) 1006900 2872 10 0.4
Methyl octacosanoate (C28) 1061700 3071 10 0.4
Methyl triacontanoate (C30) 1113100 3270 10 0.4

 aUse highest‐quality reference compounds from Sigma‐Aldrich.
 bHigher concentrations of methionine and aspartic acid are used to obtain clear spectra and peaks for manual inspection in the quality control step.
Table 0.4.2   MaterialsCompounds in External Reference QC MixPreparation and Use of FAME Mixture for Internal Standards

Compound a Retention time (sec) Solvent Weight (mg)
Pyruvate 6.74 Water 10.00
Alanine 7.53 Water 10.00
Valine 9.16 Water 10.00
Serine 9.74 Water 10.00
Nicotinic acid 10.258 Water 10.00
Succinic acid 10.52 Water 10.00
Methionine 11.82 Water 20.00 b
Aspartic acid 12 Solution A 20.00 b
4‐Hydroxyproline 12.62 Water 10.00
Salicylic acid 13.089 Water 10.00
Glutamic acid 13.37 Solution A 10.00
Creatinine 13.66 Water 10.00
α‐Ketoglutaric acid 13.86 Water 10.00
N‐Acetylaspartic acid 14.8 Water 10.00
Asparagine 14.97 Water 10.00
Putrescine 15.77 Water 10.00
Shikimic acid 16.493 Water 10.00
Citric acid 16.63 Water 10.00
Lysine 16.975 Water 10.00
D‐(+)‐Glucose 17.47 Water 10.00
Glucose‐6‐phosphate 21.381 Water 10.00
Arachidic acid 22.364 Chloroform 10.00
Serotonin 22.51 Methanol 10.00
Adenosine 23.862 Solution A 10.00
Sucrose 23.95 Water 10.00
Chlorogenic acid 26.39 Methanol 10.00
α‐Tocopherol 27.397 Chloroform 10.00
Cholesterol 27.528 Chloroform 10.00
Compound Fiehn retention index value Kovats retention index value Amount (mg) Concentration (mg/ml)
Methyl caprylate/octanoate (C08) 262320 1083 20 0.8
Methyl pelargonate/nonanoate (C09) 323120 1183 20 0.8
Methyl caprate/decanoate (C10) 381020 1282 20 0.8
Methyl laurate/dodecanoate (C12) 487220 1481 20 0.8
Methyl myristate/tetradecanoate (C14) 582620 1680 20 0.8
Methyl palmitate/hexadecanoate (C16) 668720 1878 20 0.8
Methyl stearate/octadecanoate (C18) 747420 2077 10 0.4
Methyl arachidate/icosanoate (C20) 819620 2276 10 0.4
Methyl behenate/docosanoate (C22) 886620 2475 10 0.4
Methyl tetracosanoate (C24) 948820 2674 10 0.4
Methyl hexacosanoate (C26) 1006900 2872 10 0.4
Methyl octacosanoate (C28) 1061700 3071 10 0.4
Methyl triacontanoate (C30) 1113100 3270 10 0.4

Basic Protocol 2: GC‐MS Data Acquisition for Metabolome Analysis

  Materials
  • Samples (see protocol 1)
  • Biological dummy samples, e.g., commercial blood plasma not needed for any standardization or QC purpose (prepared as for samples)
  • Ethyl acetate, LCMS‐grade
  • Helium, 5.0 grade
  • Perfluorotributylamine (FC43)
  • GC‐MS consumables such as nuts, ferrules, multi‐baffled glass liners, septa, column cutters (Restek)
  • Mass spectrometer, e.g.:
    • 6890 or 7890 Agilent GC with Leco Pegasus IV TOF MS
    • 6890 or 7890 Agilent GC with Agilent 5977 A quadrupole MS
    • 7890 Agilent GC with Agilent 7200 quadrupole/TOF MS
  • Autosampler, e.g.:
    • Gerstel Automatic Liner EXchange (ALEX) system with multipurpose autosampler system (MPS2) and cold injection system (CIS)
    • Agilent 7693 autosampler
  • Columns, e.g.:
    • Restek 95% dimethyl/5% diphenyl polysiloxane RTX‐5MS column (30‐m length, 0.25‐mm internal diameter, 0.25‐μm film) with 10‐m empty Restek guard column
    • Agilent 95% dimethyl/5% diphenyl polysiloxane J&W DB‐5MS column (30‐m length, 0.25‐mm internal diameter, 0.25‐μm film) with 10‐m empty DuraGuard guard column

Basic Protocol 3: GC‐MS Raw Data Quality Control for Metabolome Analysis

  Materials
  • For Leco GC‐TOF MS data sets
    • ChromaTOF instrument peak finding and mass spectral deconvolution software, version 4.0 or higher
    • BinBase database software (open source; https://code.google.com/p/binbase/)
  • For Agilent GC‐quadrupole MS data sets
    • Automated mass spectral deconvolution and identification system (AMDIS; from NIST)
    • SpectConnect software (developed at Georgia Tech; Styczynski et al., )
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Figures

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Literature Cited

Literature Cited
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