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.
Highlights
Pathological protein deposits have a long history as hallmarks of neurodegenerative disease
We have shown that the Spatially targeted optical microproteomics (STOMP) technique can be used to determine the proteomic composition of micron-scale microscopic features
Using amyloid plaques in Transgenic CRND8 (TgCRND8) mice as a test subject, we demonstrated that STOMP is capable of determining the protein composition of pathological deposits in neurodegenerative disease
Summary
Pathological protein deposits have a long history as hallmarks of neurodegenerative disease. Methods used to identify these deposits include the silver stain introduced by Golgi (1873), and Virchow’s iodine–sulfuric acid stain for starch (Virchow, 1854) that led to the coining of the term amyloid (Virchow, 1855). More specialized stains and labels have emerged which have begun to probe the structure and composition of pathological protein deposits. The stains Congo red and thioflavin S (ThS) were discovered later and remain in use (reviewed by Westermark (Sipe and Westermark, 2005) and Tanskanen (Tanskanen, 2013)), identifying deposits containing a particular structural motif: amyloid β-pleated sheets. Beyond mere detection lies comprehensive identification of the protein components of these deposits; biochemical analyses of the deposits have
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