Optimizing 2D gas chromatography mass spectrometry for robust tissue, serum and urine metabolite profiling.
Two-dimensional gas chromatography mass spectrometry (GCxGC-MS) is utilized to an increasing extent in biomedical metabolomics. Here, we established and adapted metabolite extraction and derivatization protocols for cell/tissue biopsy, serum and urine samples according to their individual properties. GCxGC-MS analysis revealed detection of ~600 molecular features from which 165 were characterized representing different classes such as amino acids, fatty acids, lipids, carbohydrates, nucleotides and small polar components of glycolysis and the Krebs cycle using electron impact (EI) spectrum matching and validation using external standard compounds. Advantages of two-dimensional gas chromatography based resolution were demonstrated by optimizing gradient length and separation through modulation between the first and second column, leading to a marked increase in metabolite identification due to improved separation as exemplified for lactate versus pyruvate, talopyranose versus methyl palmitate and inosine versus docosahexaenoic acid. Our results demonstrate that GCxGC-MS represents a robust metabolomics platform for discovery and targeted studies that can be used with samples derived from the clinic.
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
Zhanru
Z
Yu
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
Honglei
H
Huang
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
Alexander
A
Reim
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
Philip D
PD
Charles
Shimadzu UK Limited, Mill Court, Featherstone Road Wolverton, Mill South, Milton Keynes MK12 5RD, UK.
Alan
A
Northage
Shimadzu UK Limited, Mill Court, Featherstone Road Wolverton, Mill South, Milton Keynes MK12 5RD, UK.
Dianne
D
Jackson
Shimadzu UK Limited, Mill Court, Featherstone Road Wolverton, Mill South, Milton Keynes MK12 5RD, UK.
Ian
I
Parry
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK. Electronic address: benedikt.kessler@ndm.ox.ac.uk.
Benedikt M
BM
Kessler
D015415:Biomarkers / Q000032:analysis*; D002477:Cells / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods* / Q000592:standards*; D006801:Humans; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D044967:Serum / Q000378:metabolism*; D016482:Urinalysis / Q000379:methods*
D015415:Biomarkers
Bio fluid; Electron impact spectrum; Mass spectrometry; Metabolomics; Tissue cell extract; Two dimensional gas chromatography