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A complete workflow for high-resolution spectral-stitching nanoelectrospray direct-infusion mass-spectrometry-based metabolomics and lipidomics

This article has been updated

Abstract

Metabolomic and lipidomic studies measure and discover metabolic and lipid profiles in biological samples, enabling a better understanding of the metabolism of specific biological phenotypes. Accurate biological interpretations require high analytical reproducibility and sensitivity, and standardized and transparent data processing. Here we describe a complete workflow for nanoelectrospray ionization (nESI) direct-infusion mass spectrometry (DIMS) metabolomics and lipidomics. After metabolite and lipid extraction from tissues and biofluids, samples are directly infused into a high-resolution mass spectrometer (e.g., Orbitrap) using a chip-based nESI sample delivery system. nESI functions to minimize ionization suppression or enhancement effects as compared with standard electrospray ionization (ESI). Our analytical technique—named spectral stitching—measures data as several overlapping mass-to-charge (m/z) windows that are subsequently 'stitched' together, creating a complete mass spectrum. This considerably increases the dynamic range and detection sensitivity—about a fivefold increase in peak detection—as compared with the collection of DIMS data as a single wide mass-to-charge (m/z ratio) window. Data processing, statistical analysis and metabolite annotation are executed as a workflow within the user-friendly, transparent and freely available Galaxy platform (galaxyproject.org). Generated data have high mass accuracy that enables molecular formulae peak annotations. The workflow is compatible with any sample-extraction method; in this protocol, the examples are extracted using a biphasic method, with methanol, chloroform and water as the solvents. The complete workflow is reproducible, rapid and automated, which enables cost-effective analysis of >10,000 samples per year, making it ideal for high-throughput metabolomics and lipidomics screening—e.g., for clinical phenotyping, drug screening and toxicity testing.

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Figure 1: Flowchart showing the full spectral-stitching nESI DIMS workflow.
Figure 2: Schematic of the spectral-stitching nESI DIMS method for application on the Q Exactive Hybrid Quadrupole Orbitrap mass spectrometer.
Figure 3: Recommended arrangement of samples in a 96-well plate for a spectral-stitching nESI DIMS experiment.
Figure 4: Detection sensitivity of the spectral-stitching nESI DIMS method (blue) is superior to standard full-scan analysis (dark orange).
Figure 5: Analysis of a human leukemia cell line (HL60) lipid extract by the spectral-stitching nESI DIMS method.
Figure 6
Figure 7: Intensity measurements by the spectral-stitching nESI DIMS method are consistent with NMR spectroscopic measurements.

Change history

  • 09 February 2017

    In the version of this article initially published online, the page numbers were assigned incorrectly as 255–273. The correct page numbers are 310–328. The error has been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

A.D.S. was funded by a Bloodwise programme grant (no. 13028) awarded to F.L. Khanim and M.T. Drayson. R.J.M.W., M.R.V., J.E. and M.R.J. were funded by the UK Natural Environment Research Council (grants NE/I008314/1, NE/J017442/1, NE/K011294/1 and R8-H10-61), and R.J.M.W. and M.R.V. were funded by the UK Biotechnology and Biological Sciences Research Council (grant BB/M019985/1). M.R.J. thanks Thermo Fisher Scientific for CASE funding. The LTQ FT Ultra used in this research was obtained through the Birmingham Science City Translational Medicine: Experimental Medicine Network of Excellence project, with support from Advantage West Midlands (AWM). We thank N. Taylor (University of Birmingham, UK) and collaborators (J. Gunn, Laurentian University, Canada; and J. McGeer, Wilfrid Laurier University, Canada), who kindly provided us with experimental data for Figure 6. We thank D. Broadhurst (University of Alberta) for ongoing development and support for the batch-correction algorithm.

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A.D.S. and R.J.M.W. codeveloped the analytical methods and computational workflow, acquired data and wrote the manuscript. J.E. contributed to developing the computational workflow and writing of the manuscript. M.R.J. contributed to developing the analytical methods and acquired data. M.R.V. codeveloped the analytical methods and computational workflows, edited the manuscript and was the academic lead.

Corresponding author

Correspondence to Mark R Viant.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods 1

Q Exactive instrument method files. (ZIP 309 kb)

Supplementary Methods 2

Orbitrap Elite instrument method files. (ZIP 47 kb)

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Southam, A., Weber, R., Engel, J. et al. A complete workflow for high-resolution spectral-stitching nanoelectrospray direct-infusion mass-spectrometry-based metabolomics and lipidomics. Nat Protoc 12, 310–328 (2017). https://doi.org/10.1038/nprot.2016.156

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