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Rapid ex vivo molecular fingerprinting of biofluids using laser-assisted rapid evaporative ionization mass spectrometry

Nature Protocols volume 16, pages 4327–4354 (2021)Cite this article

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Abstract

Of the many metabolites involved in any clinical condition, only a narrow range of biomarkers is currently being used in the clinical setting. A key to personalized medicine would be to extend this range. Metabolic fingerprinting provides a more comprehensive insight, but many methods used for metabolomics analysis are too complex and time-consuming to be diagnostically useful. Here, a rapid evaporative ionization mass spectrometry (REIMS) system for direct ex vivo real-time analysis of biofluids with minor sample pretreatment is detailed. The REIMS can be linked to various laser wavelength systems (such as optical parametric oscillator or CO2 laser) and with automation for high-throughput analysis. Laser-induced sample evaporation occurs within seconds through radiative heating with the plume guided to the MS instrument. The presented procedure includes (i) laser setup with automation, (ii) analysis of biofluids (blood/urine/stool/saliva/sputum/breast milk) and (iii) data analysis. We provide the optimal settings for biofluid analysis and quality control, enabling sensitive, precise and robust analysis. Using the automated setup, 96 samples can be analyzed in ~35–40 min per ionization mode, with no intervention required. Metabolic fingerprints are made up of 2,000–4,000 features, for which relative quantification can be achieved at high repeatability when total ion current normalization is applied. With saliva and feces as example matrices, >70% of features had a coefficient of variance ≤30%. However, to achieve acceptable long-term reproducibility, additional normalizations by, e.g., LOESS are recommended, especially for positive ionization.

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Fig. 1: A schematic representation of the LA-REIMS workflow.
Fig. 2: A flowchart of a standard LA-REIMS biofluid analysis experiment.
Fig. 3: Typical burns for LA-REIMS analysis.
Fig. 4: Close-up of the motorized automated sampling area and its connection to the REIMS source.
Fig. 5: Typical mass spectra as obtained by LA-REIMS analysis of human biofluids.
Fig. 6: Typical m/z profiles as obtained by LA-REIMS analysis of human biofluids.
Fig. 7: Multivariate modeling of pathophysiological state based on LA-REIMS fingerprints.

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Data availability

Datasets relevant to our published supporting primary papers can be made available from the corresponding author upon reasonable request. The source data for figures are publicly available in the Figshare repository: Fig. 3, https://doi.org/10.6084/m9.figshare.14258423.v1; Fig. 5, https://doi.org/10.6084/m9.figshare.14258438.v1; Fig. 6 https://doi.org/10.6084/m9.figshare.14258459.v2.

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Acknowledgements

This work was supported by VLAIO (grant number IM 150740 – A16/OC/0006), BOF GOA (grant number 2017/000102), BOF (grant number 01J07519), FWO Hercules (grant number AUG/17/09), FWO (grant numbers 1S57920N and 1S49020N) and ERC FWO Runner-up (grant number G0G0119N). The Laboratory of Chemical Analysis research group is part of the Ghent University expertise centre MSsmall.

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Author notes

  1. These authors contributed equally: Vera Plekhova, Lieven Van Meulebroek.

Authors and Affiliations

  1. Laboratory of Chemical Analysis, Ghent University, Merelbeke, Belgium

    Vera Plekhova, Lieven Van Meulebroek, Marilyn De Graeve, Margot De Spiegeleer, Ellen De Paepe, Emma Van de Walle & Lynn Vanhaecke

  2. ProDigest BV, Zwijnaarde, Belgium

    Lieven Van Meulebroek

  3. Department of Surgery and Cancer, Imperial College London, London, UK

    Alvaro Perdones-Montero & Zoltan Takats

  4. School of Biological Sciences, Institute for Global Food Security, Queen’s University Belfast, Belfast, Northern Ireland, UK

    Simon J. S. Cameron & Lynn Vanhaecke

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Contributions

V.P., L.V.M., M.D.G., M.D.S., E.D.P. and S.C. wrote the original draft of the manuscript. V.P., L.V.M., E.V.d.W. and A.P. carried out the experiments. M.D.G. and A.P. performed the data analysis. L.V.M., E.D.P. and S.C. collected the samples. Z.T. and S.C. developed the technique. L.V., Z.T. and S.C. supervised the project and provided the funding. L.V., M.D.S. and L.V.M. performed the proofreading and correction of the manuscript.

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Correspondence to Lynn Vanhaecke.

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Key references using this protocol

Cameron, S. et al. Anal. Chem. 91, 13488 (2019): https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b02358

Wijnant, K. et al. Anal. Chem. 92, 5116 (2020): https://pubs.acs.org/doi/10.1021/acs.analchem.9b05598

Van Meulebroek, L. et al. Talanta 217, 121043 (2020): https://www.sciencedirect.com/science/article/pii/S0039914020303349

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Plekhova, V., Van Meulebroek, L., De Graeve, M. et al. Rapid ex vivo molecular fingerprinting of biofluids using laser-assisted rapid evaporative ionization mass spectrometry. Nat Protoc 16, 4327–4354 (2021). https://doi.org/10.1038/s41596-021-00580-8

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