Abstract 5638: Total-sync ultra-content microscopic opto-biotinylation enables high-sensitivity hypothesis-free subcellular protein discovery

High-sensitivity hypothesis-free subcellular proteomics is challenging due to the limited sensitivity of mass spectrometry and the lack of amplification tools for proteins. Without such technology, it is not possible to discover proteins at specific locations of interest in cells or tissue samples. Here, we introduce a total-sync ultra-content microscopic system termed MicroscoopTM that integrates microscopy, optics, FPGA-based mechatronics, photochemistry, and deep learning or computer vision to enable high-content in situ photolabeling. MicroscoopTM photolabels proteins at user defined regions of interests (ROIs) under a microscope utilizing directed photochemistry in one field of view (FOV) at a time for tens of thousands of FOVs with similar morphological features. With this platform, we are able to photolabel proteins with biotin probes in cellular organelles, granules or cell-cell contact surfaces with a high precision at nanoscale resolution, and obtain sufficient amount of biotinylated proteins for mass spectrometry. We made a robust demonstration in the proteome mapping of human cellular nucleus from single-shot experiment to >1000 nuclear protein identification with > 90% specificity. Further data analysis revealed identification of a hundred of low protein copy number proteins and a high coverage of nuclear complexes. In proteome mapping of the nucleolus, we ranked proteins by order of abundance and revealed that 97 out of the top 100 proteins were annotated as nucleolar proteins. Unexpectedly, in mapping the stress granule (SG) proteome, a relatively low SG specificity (74%) were found in the top 50 abundant proteins, therefore we further characterize the proteins that have no prior SG annotation by immunostaining. Nine out of the thirteen unexplored proteins including PDLIM7, EIF3CL, YWHAE, RPSA, UGDH, DDX17, ANLN, PSMA6, and MCM2 were found to have SG patterns and co-localized with SG marker (G3BP1), raising our top 50 SG specificity to up to 92%. Together, our total-sync ultra-content microscopic platform enables hypothesis-free, de novo subcellular proteome mapping at user defined ROIs with high sensitivity and specificity, thereby broadly benefits the cell biology field in finding novel proteins or biomarkers. Citation Format: Jung-Chi Liao, Chih-Wei Chang, Yi-De Chen, Chantal Hoi Yin Cheung, Chia-Wen Chung, Hsiao-Jen Chang, Yong-Da Sie, You-Pi Liu, Yu-Chih Lin, Hsiang-Ju Kai, Weng Man Chong, Hsin-Yi Wu. Total-sync ultra-content microscopi