The exact composition of proteins at a specific subcellular region is important to know in order to understand their underlying physiological roles for cellular functions and potentially shed light on the molecular mechanism of associated diseases. Currently, spatial proteomics is achieved through affinity-based biochemical assays associated a bait protein. However, this bait protein may be present in multiple locations, resulting in unwanted background in the obtained proteome. Mass spectrometry imaging is another approach for spatial proteomics, but its resolution cannot reach a subcellular level. Here we develop a novel spatially resolved subcellular proteomic mapping technology based on user-defined characteristics of cellular images, where the labeling area is determined by image processing of specified characteristics. 3D photochemical labeling is achieved by utilizing two-photon microscope illumination to trigger the photoredox reaction of Ru(bpy)32+, a powerful photocatalyst which can drive desthiobiotin-tyramide to bind to the proteins within the focal spot. The labeled proteins can be purified with streptavidin enrichment after solubilizing the fixed cell sample. Furthermore, by automating high-throughput image-guided two-photon labeling and optimizing sample preparation protocols of low abundance proteins for mass spectrometry, we demonstrate that this technology enables spatially specific isolation of proteins from hundreds of thousands of individual cells within a few days of automated two-photon labeling. Together, this unprecedented image-based subcellular proteomic mapping possesses an extensive potential in broad biological applications when protein identification of a specific region under a microscope is desired.

Image-guided subcellular proteomic mapping using Ru(bpy)32+ catalyzed two-photon labeling

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.