Transforming Spatial Proteomics via Simultaneous Multiplexed Imaging at Single-Protein Resolution - PROJECT SUMMARY Understanding biological processes requires not only measuring how much of a protein is present, but also knowing where each protein is located, how it is organized, and with whom it interacts. Existing mass spectrometry–based proteomics methods provide comprehensive molecular profiling but lack the spatial resolution necessary to resolve nanoscale protein organization. Imaging-based spatial proteomics techniques such as imaging mass spectrometry, multiplexed ion beam imaging, and CODEX can profile up to 100 proteins with subcellular resolution, but they are limited to 200–500 nm resolution and cannot visualize nanoscale protein assemblies at the molecular scale. Single-molecule localization microscopy (SMLM) achieves nanometer precision, enabling visualization of proteins at the scale of individual molecules. Among these methods, DNA-PAINT provides theoretically unlimited multiplexing but suffers from slow acquisition, sequential imaging requirements, sample damage from repeated washing, system instability during multi-round imaging, and high background in thick samples. This project introduces Simul-Proteomics, a next-generation spatial proteomics platform that enables high-throughput, simultaneous multiplexed imaging at single-protein resolution. By integrating transformative spectral phasor analysis, spectrally engineered probes such as quantum dots, and light sheet illumination, Simul-Proteomics directly overcomes the limitations of DNA-PAINT. This approach eliminates sequential readout, minimizes sample perturbation, and improves speed by more than 100-fold for up to 60-plexed imaging, enabling quantitative nanoscale mapping of protein distributions and interactions in cells, tissues, and small organisms in a single round. The proposed research will deliver a transformative technology platform capable of resolving nanoscale protein organization in complex biological systems with unprecedented throughput. This will open the door to answering fundamental biological questions about protein assemblies, signaling networks, and spatial regulation in health and disease. By achieving high- throughput single-protein spatial proteomics, the project has the potential to impact diverse fields including cancer biology, neurobiology, and developmental biology, while also laying the groundwork for future translational applications in diagnostics and therapeutics.
