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Determining composition of micron-scale protein deposits in neurodegenerative disease by spatially targeted optical
Kevin C Hadley1, Rishi Rakhit2, Hongbo Guo3
1Department of Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, Canada.
Abstract:
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.
Insights
Spatially targeted optical microproteomics (STOMP) enables detailed protein analysis in specific tissue regions without genetic modification. This novel method identified known and new proteins in Alzheimer
Area of Science:
- Proteomics
- Biochemistry
- Cell Biology
Background:
- Investigating protein composition in specific tissue regions is crucial for understanding disease mechanisms.
- Existing proteomics techniques often lack spatial resolution or require genetic manipulation.
- Analyzing micron-scale regions of interest (ROIs) in mammalian tissue presents significant technical challenges.
Purpose of the Study:
- To introduce Spatially Targeted Optical Microproteomics (STOMP), a novel technique for high-resolution spatial proteomics.
- To demonstrate the applicability of STOMP for identifying protein constituents within specific ROIs in biological samples.
- To validate STOMP's utility by analyzing amyloid plaques in Alzheimer's disease models.
Main Methods:
- STOMP utilizes photo-tagging of proteins within visualized ROIs in fixed tissue specimens.
- Confocal and two-photon excitation microscopy enable precise photo-tag coupling to proteins at sub-micron resolution.
- Photo-tagged proteins are isolated after tissue solubilization and identified using mass spectrometry.
Main Results:
- STOMP successfully identified known amyloid plaque constituents in Alzheimer's disease models.
- The technique revealed novel protein components associated with amyloid plaques.
- STOMP demonstrated high spatial resolution (0.67 µm xy, 1.48 µm axial) in fixed tissue.
Conclusions:
- STOMP is a versatile and powerful proteomics technique for interrogating micron-scale ROIs in diverse biological samples.
- This method offers a non-genetic approach to spatial proteomics, advancing the study of tissue composition.
- STOMP has significant potential for applications in disease research, diagnostics, and drug discovery.
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