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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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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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.
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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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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
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Supplementary Information
Supplementary Figs. 1–3, Supplementary Tables 1–3 and Supplementary References.
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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DOI: https://doi.org/10.1038/s41596-021-00538-w
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