Determining composition of micron-scale protein deposits in neurodegenerative disease by spatially targeted optical microproteomics

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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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Kevin C Hadley

Department of Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada

Competing interests
The authors declare that no competing interests exist.
Rishi Rakhit

Department of Chemical and Systems Biology, Stanford University, Stanford, United States

Competing interests
The authors declare that no competing interests exist.
Hongbo Guo

The Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular & Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, Canada

Competing interests
The authors declare that no competing interests exist.
Yulong Sun

Department of Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada

Competing interests
The authors declare that no competing interests exist.
James EN Jonkman

Advanced Optical Microscopy Facility, Princess Margaret Cancer Centre, University Health Network, Toronto, Canada

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The authors declare that no competing interests exist.
Joanne McLaurin

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

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The authors declare that no competing interests exist.
Lili-Naz Hazrati

Tanz Centre for Research in Neurodegenerative Diseases, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

Competing interests
The authors declare that no competing interests exist.
Andrew Emili

The Banting and Best Department of Medical Research, Terrence Donnelly Centre for Cellular & Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, Canada

Competing interests
The authors declare that no competing interests exist.
Avijit Chakrabartty

Departments of Biochemistry and Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada

For correspondence
chakrab@uhnresearch.ca

Competing interests
The authors declare that no competing interests exist.

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.

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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.