Determining composition of micron-scale protein deposits in neurodegenerative disease by spatially targeted optical microproteomics
- University of Toronto, Canada
- Stanford University, United States
- University Health Network, Canada
Abstract
Spatially targeted optical microproteomics (STOMP) is a novel proteomics technique for interrogating micron-scale regions of interest (ROI) in mammalian tissue, with no requirement for genetic manipulation. Methanol or formalin fixed specimens are stained with fluorescent dyes or antibodies to visualize ROIs, then soaked in solutions containing the photo-tag: 4-benzoylbenzyl-glycyl-hexahistidine. Confocal imaging along with two photon excitation are used to covalently couple photo-tags to all proteins within each ROI, to a resolution of 0.67 µm in the xy-plane and 1.48 µm axially. After tissue solubilization, photo-tagged proteins are isolated and identified by mass spectrometry. As a test case, we examined amyloid plaques in an Alzheimer's disease (AD) mouse model and a postmortem AD case, confirming known plaque constituents and discovering new ones. STOMP can be applied to various biological samples including cell lines, primary cell cultures, ex vivo specimens, biopsy samples and fixed postmortem tissue.
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Ethics
Animal experimentation: This study was performed in strict accordance with the University of Toronto Animal Care Committee Guidelines.
Human subjects: The work presented was performed in compliance with recognized international standards, including the International Conference on Harmonization (ICH), the Council for International Organizations of Medical Sciences (CIOMS) and the principles of the Declaration of Helsinki. Use of human tissue was in accordance with the University Health Network Research Ethics Board. The Human brain samples were collected as part of the Canadian Brain Tissue Bank (CBTB). At the time of collection, informed consent was obtained.
Copyright
© 2015, Hadley et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Determining composition of micron-scale protein deposits in neurodegenerative disease by spatially targeted optical microproteomics
eLife 4:e09579.
https://doi.org/10.7554/eLife.09579
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