Proteome-Driven Phenotyping of Identified Single Neurons in Intact Brain Tissue by Aspiration Patch Proteomics

Single-cell proteomics has advanced rapidly, but direct proteome measurements from identified neurons in intact brain tissue remain difficult because most workflows require cell isolation and recent patch-based studies have emphasized whole-soma retrieval. Here we show that aspiration-based patch proteomics enables deep proteome profiling of identified single neurons directly in acute mouse brain slices. We combined fluorescence-guided patch-clamp microsampling, minimal-loss bottom-up proteomics, and high-sensitivity capillary electrophoresis-timsTOF mass spectrometry to analyze partial somal aspirates from dopaminergic, parvalbumin, and serotonergic neurons in situ. The workflow identified more than 1,000 proteins from single-neuron samples under optimized conditions while consuming only about 0.25% of the processed digest per analysis. These proteomes were sufficient to separate biological replicates by neuronal phenotype, distinguish neuronal subtypes on the basis of protein expression alone, and define a conserved somal proteome shared across neuronal classes. Our results establish that controlled aspiration of partial somal material can support proteome-driven phenotyping without whole-soma retrieval, cell dissociation, or loss of native tissue context. Aspiration patch proteomics therefore provides an accessible route for subtype-level proteome phenotyping in intact brain tissue.