Aggregate Deep Visual Proteomics reveals selective organization of α-synuclein inclusions

α-Synuclein inclusions, the lesions typical of Parkinson's disease, have been interpreted as protective deposits, toxic species or failed degradation products, each predicting a different composition and correlating that enrichment with neuron dysfunction and eventual degeneration. Distinguishing them requires measuring what an inclusion concentrates relative to the cell that built it. Here we developed Aggregate Deep Visual Proteomics, which uses ultrasensitive mass spectrometry to profile laser-microdissected inclusions against the remainder of the same cell or a matched host. In HEK293 cells, iPSC-derived dopaminergic neurons and post-mortem patient brain, each inclusion concentrated a selective subset of its cell's proteome, independent of abundance and dominated by membrane, in neurons largely presynaptic. Inclusions took up whole assemblies, with subunits moving together in 421 of 438 complexes, and the V-ATPase alone was enriched in all three systems. Degradation cargo stayed with the receptors that route it, consistent with sequestration and failed degradation acting together. Neuritic and somatic inclusions differed, the former rich in UBQLN2 and the latter in intermediate filaments. In the cell line, phosphorylated α-synuclein was tenfold higher in the inclusion than in the rest of the cell, with about one third of the rise already present before an inclusion was visible. Measured against its host, the composition of an inclusion becomes a readout of how it formed and a source of candidate markers and therapeutic targets.