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Poster Presentations
Day 4, June 25（Wed.）
Room P (Maesato East, Foyer, Ocean Wing)
- 4P-PM-13
High multiplexity imaging proteomics via tissue expansion
(HKBU)
oXin Diao, Jianing Wang, Zongwei Cai
Spatial proteomics holds transformative potential in biomedical research, providing ability to map protein localization and interactions within tissues microenvironment, advancing understanding of complex biological processes. However, conventional fluorescence-based antibody imaging methods face limitations in multiplexing capacity due to spectral overlaps and autofluorescence. While metal-labelled antibodies have enabled multiplexed imaging of up to 40 proteins at subcellular resolution, scalability remains constrained by the finite availability of metal isotopes. Here, we present a novel workflow that integrates tissue expansion with photocleavable Mass-Tag (PCMT) antibody labeling and MALDI-MSI to achieve highly multiplexed spatial proteomics. Tissues were embedded in a swellable hydrogel, enabling isotropic expansion to 10-fold while preserving antigen integrity. Following expansion, PCMT antibodies were conjugated to the tissue and imaged with MALDI-MSI. This approach achieved subcellular spatial resolution and demonstrated over 60-plex capability in mouse tissues, surpassing current metal-based multiplexing limits. Crucially, PCMT antibody decouples multiplexing capacity from the limitations of labels, enabling theoretically unlimited protein targets to be analyzed in a single experiment. By overcoming the bottlenecks of spectral overlap and label scarcity, our workflow establishes a scalable platform for subcellular resolution, hyper-multiplexed spatial proteomics.