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Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging

Nature Protocols volume 16, pages 3298–3321 (2021)Cite this article

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Abstract

Molecule-specific techniques such as MALDI and desorption electrospray ionization mass spectrometry imaging enable direct and simultaneous mapping of biomolecules in tissue sections in a single experiment. However, neurotransmitter imaging in the complex environment of biological samples remains challenging. Our covalent charge-tagging approach using on-tissue chemical derivatization of primary and secondary amines and phenolic hydroxyls enables comprehensive mapping of neurotransmitter networks. Here, we present robust and easy-to-use chemical derivatization protocols that facilitate quantitative and simultaneous molecular imaging of complete neurotransmitter systems and drugs in diverse biological tissue sections with high lateral resolution. This is currently not possible with any other imaging technique. The protocol, using fluoromethylpyridinium and pyrylium reagents, describes all steps from tissue preparation (~1 h), chemical derivatization (1–2 h), data collection (timing depends on the number of samples and lateral resolution) and data analysis and interpretation. The specificity of the chemical reaction can also help users identify unknown chemical identities. Our protocol can reveal the cellular locations in which signaling molecules act and thus shed light on the complex responses that occur after the administration of drugs or during the course of a disease.

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Fig. 1: Optimization of the FMP-10 matrix application protocol for MALDI-MSI.
Fig. 2: Detection of multiple neurotransmitters by MALDI-MSI using DPP and FMP-10.
Fig. 3: MALDI-MSI analysis of neurotransmitters, metabolites and amino acids facilitated by on-tissue FMP-10 derivatization.
Fig. 4: High-resolution MALDI-MSI analysis of a rat brain tissue section after on-tissue derivatization with FMP-10.
Fig. 5: MALDI-MSI analysis of neurotransmitters facilitated by on-tissue derivatization with DPP.
Fig. 6: DESI-MSI analysis of neurotransmitters facilitated by on-tissue derivatization by FMP-10.

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Data availability

The raw datasets for both MALDI and DESI-MSI experiments are too large to be publicly shared but are available for research purposes from the corresponding author upon reasonable request. Source data for Table 1 and Supplementary Table 1 are provided as Supplementary Data.

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Acknowledgements

This work was financially supported by the Swedish Research Council (Medicine and Health Grant 2018–03320 and Natural and Engineering Science Grants 2018–05501 and 2018-05133), the Swedish Brain Foundation (FO2018-0292), the Swedish Foundation for Strategic Research (RIF14-0078) and the Science for Life Laboratory (SciLifeLab).

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Author notes

  1. These authors contributed equally: Reza Shariatgorji, Anna Nilsson.

Authors and Affiliations

  1. Department of Pharmaceutical Biosciences, Medical Mass Spectrometry Imaging, Uppsala University, Uppsala, Sweden

    Reza Shariatgorji, Anna Nilsson, Elva Fridjonsdottir & Per E. Andrén

  2. Science for Life Laboratory, Spatial Mass Spectrometry, Uppsala University, Uppsala, Sweden

    Reza Shariatgorji, Anna Nilsson & Per E. Andrén

  3. Imaging & Data Analytics, Clinical Pharmacology and Safety Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK

    Nicole Strittmatter, Andreas Dannhorn & Richard J. A. Goodwin

  4. Department of Clinical Neuroscience, Section of Neurology, Karolinska Institutet, Stockholm, Sweden

    Per Svenningsson

  5. Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden

    Luke R. Odell

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Contributions

R.S. and A.N. conceived the methodology; designed experiments; acquired, analyzed and interpreted data; and wrote the manuscript. E.F. analyzed data and wrote part of the manuscript. N.S. and A.D. designed experiments; acquired, analyzed and interpreted data; and wrote part of the manuscript. P.S. provided tissue samples, supervised animal experiments and edited the manuscript. R.J.A.G. designed experiments, analyzed and interpreted data and wrote part of the manuscript. L.R.O. conceived the methodology, designed experiments, synthesized the reactive matrices, interpreted data and edited the manuscript. P.E.A. conceived the methodology, designed experiments, interpreted data, wrote the manuscript and is principal investigator for the grants that fund this research.

Corresponding author

Correspondence to Per E. Andrén.

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Competing interests

N.S, A.D. and R.J.A.G. are full-time salaried employees, own stocks of AstraZeneca and performed this study as part of their regular duties. None of the authors received any form of royalty or further financial compensation from AstraZeneca for publishing these protocols. L.R.O., A.N., R. S. and P.E.A. are co-founders and shareholders in Tag-ON AB and have filed a patent application ‘Reactive desorption and/or laser ablation ionization matrices and use thereof’, no. PCT/SE2019/050197 (patent pending).

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Peer review information Nature Protocols thanks Axel Karl Walch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Shariatgorji, M. et al. Nat. Methods 16, 1021–1028 (2019): https://doi.org/10.1038/s41592-019-0551-3

Shariatgorji, M. et al. Neuron 84, 697–707 (2014): https://doi.org/10.1016/j.neuron.2014.10.011

Shariatgorji, M. et al. Neuroimage 136, 129–38 (2016): https://doi.org/10.1016/j.neuroimage.2016.05.004

Supplementary information

Supplementary Information

Supplementary Figs. 1–3, Supplementary Tables 1–3 and Supplementary References.

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Supplementary Data

Mass accuracy calculations, red phosphorus mass reference table and source data for LOD calculations

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Shariatgorji, R., Nilsson, A., Fridjonsdottir, E. et al. Spatial visualization of comprehensive brain neurotransmitter systems and neuroactive substances by selective in situ chemical derivatization mass spectrometry imaging. Nat Protoc 16, 3298–3321 (2021). https://doi.org/10.1038/s41596-021-00538-w

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