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We introduce RCAmp-scWB, a novel post-separation amplification strategy for single-cell western blotting that performs rolling-circle amplification directly within a polyacrylamide gel. This method achieves a 4.0-7.8-fold steeper concentration-response slope than conventional single-cell western blotting by balancing reagent access and template confinement to preserve spatial readout. RCAmp-scWB successfully resolved distinct Vimentin and PD-L1 proteoform states in human cancer cell lines and extracellular vesicles, revealing heterogeneity not captured by total protein abundance.
Single-cell western blotting now resolves proteoform diversity, revealing cancer cell and extracellular vesicle heterogeneity previously masked by total protein abundance.
Protein abundance alone does not capture the molecular diversity generated by protein processing and post-translational modification, yet most single-cell protein assays do not resolve these proteoform states. Here, we develop RCAmp-scWB, a post-separation amplification strategy that performs rolling-circle amplification directly within the polyacrylamide gel after single-cell protein electrophoresis. Reaction-transport modeling identifies a balance between reagent access and confinement of the template-associated amplification product that supports signal amplification while retaining the electrophoretically encoded spatial readout. Using purified protein standards, RCAmp-scWB produced a 4.0-7.8-fold steeper concentration-response slope than conventional single-cell western blotting. Across six human cancer cell lines, RCAmp-scWB quantified protein abundance and resolved distinct Vimentin and PD-L1 proteoform states whose relative abundance and single-cell distributions were not captured by total protein measurements alone. Extension to individual small extracellular vesicles further revealed source-dependent shifts in Vimentin proteoform composition despite comparatively similar abundance of one major Vimentin species. By separating electrophoretic molecular discrimination from signal amplification, RCAmp-scWB enables proteoform-resolved analysis of heterogeneous cells and small extracellular vesicles.