Determining composition of micron-scale protein deposits in neurodegenerative disease by spatially targeted optical microproteomics
Kevin C Hadley,
Rishi Rakhit,
Hongbo Guo,
Yulong Sun,
James EN Jonkman,
Joanne McLaurin,
Lili-Naz Hazrati,
Andrew Emili,
Avijit Chakrabartty
Affiliations
Kevin C Hadley
Department of Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada
Rishi Rakhit
Department of Chemical and Systems Biology, Stanford University, Stanford, United States
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
Department of Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada
James EN Jonkman
Advanced Optical Microscopy Facility, Princess Margaret Cancer Centre, University Health Network, Toronto, Canada
Joanne McLaurin
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
Lili-Naz Hazrati
Tanz Centre for Research in Neurodegenerative Diseases, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada
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
Avijit Chakrabartty
Departments of Biochemistry and Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada
Spatially targeted optical microproteomics (STOMP) is a novel proteomics technique for interrogating micron-scale regions of interest (ROIs) 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 post-mortem 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 post-mortem tissue.
