SPEx: Compartment-Resolved Proteomics via Expansion Microscopy–Guided Microdissection

Cells contain different organelles and compartments that are essential for cellular function and life. These organelles and compartments need to communicate to assess cellular state in a changing environment, adapt to the new situation, and also to ensure functionality and homeostasis. Moreover, organization and communication differ between cell types. However, our knowledge about these changes is still rather scarce. Subcellular spatial proteomics aims to fill this knowledge gap. While proximity labeling techniques represent a great advance, they do not provide precise spatial resolution. To overcome this limitation, we developed SPEx ( S ubcellular spatial P roteomics coupled to Ex pansion), in which we first expand cells about 10– fold, laser micro-dissect regions of interests and then perform mass spectrometry-based proteomics on these samples. We demonstrate the effectiveness of SPEx by determining the proteome of the Golgi, the nucleus and nucleoli. Satisfyingly, we also identify novel components of these organelles. Combining inexpensive already existing technologies makes SPEx readily usable by the wider scientific community.