Spatial proteomics in neurons at single-protein resolution

Authors:

Eduard M. Unterauer, Sayedali Shetab Boushehri, Kristina Jevdokimenko, Luciano A. Masullo, Mahipal Ganji, Shama Sograte-Idrissi, Rafal Kowalewski, Sebastian Strauss, Susanne C.M. Reinhardt, Ana Perovic, Carsten Marr, Felipe Opazo, Eugenio F. Fornasiero, Ralf Jungmann

Keywords:

DNA-PAINT super-resolution microscopy multiplexing spatial-omics proteomics neuron atlas synapse synapse diversity synaptic proteins excitatory synapses inhibitory synapses neuron imaging

Abstract:

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.

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