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Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry

Abstract

Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography–mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography–MS (GC-MS) and ultraperformance liquid chromatography–MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control–based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.

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Figure 1
Figure 2: The QC-RLSC protocol for a metabolic feature detected in UPLC-MS (ES+) with signal attenuation across a given analytical batch.
Figure 3: The data preprocessing workflow for UPLC-MS data.
Figure 4: The experimental workflow followed in the HUSERMET project.
Figure 5: Typical chromatograms observed for serum.
Figure 6: Peak area data for 1-methylnicotinamide before and after QC-RLSC.
Figure 7: Gender-specific creatinine distribution.

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Acknowledgements

The human serum metabolome project (HUSERMET) is funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) (BB/C008219/1), MRC, GlaxoSmithKline and by AstraZeneca. We thank the BBSRC and the Engineering and Physical Sciences Research Council for their financial support to The Manchester Centre for Integrative Systems Biology (BB/C008219/1). W.B.D. wishes to thank the UK National Institute for Health Research for financially supporting the Manchester Biomedical Research Centre.

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Contributions

W.B.D. developed the experimental design strategy, the quality control strategy, the analytical methods and co-wrote the paper. D.B. developed the experimental design, sample scheduling and quality control strategies, and developed the QC-RSLC algorithm, performed data analysis and co-wrote the paper. P.B. and E.Z. developed the experimental design strategy and methods and acquired data. S.F.-M. and N.A. acquired data. M.B. developed the XCMS deconvolution strategy and performed data analysis. J.D.K. developed the sample stratification algorithm. A.H., J.N.H. and A.W.N. developed the experimental design strategy. I.D.W., D.B.K. and R.G. developed the experimental design strategy and co-wrote the paper.

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Correspondence to Warwick B Dunn.

Supplementary information

Supplementary Method 1

Typical analysis orders for (A) GC-ToF-MS (PDF 5 kb)

Supplementary Method 2

Typical analysis orders for (B) UPLC-ToF-MS (PDF 5 kb)

Supplementary Method 3

Typical analysis orders for (C) UPLC-LTQ/Orbitrap-MS (PDF 6 kb)

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Dunn, W., Broadhurst, D., Begley, P. et al. Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nat Protoc 6, 1060–1083 (2011). https://doi.org/10.1038/nprot.2011.335

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