Spatial proteomics in neurons at single-protein resolution

To fully understand biological processes and functions, it is necessary to reveal the molecular heterogeneity of cells and even subcellular assemblies by gaining access to the location and interaction of all biomolecules. The study of protein arrangements has seen significant advancements through super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of spatial proteomics. Here, we introduce Secondary label-based Unlimited Multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method capable of achieving virtually unlimited multiplexing at better than 15 nm spatial resolution. Using SUM-PAINT, we generated the most extensive multiprotein dataset to date at single-protein spatial resolution, comprising up to 30 distinct protein targets in parallel and adapted omics-inspired analysis workflows to explore these feature-rich datasets. Remarkably, by examining the multiplexed protein content of almost 900 individual synapses at single-protein resolution, we revealed the complexity of synaptic heterogeneity, ultimately leading to the discovery of a new synapse type. This work provides not only a feature-rich resource for researchers, but also an integrated data acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution, paving the way for ‘Localizomics’. ### Competing Interest Statement E.M.U., M.G. and R.J. have filed a patent on the method published in this paper. F.O. is a shareholder of NanoTag Biotechnologies GmbH. The other authors declare no competing financial interests.