Single-cell and spatial proteomics workflows for in-depth tissue mapping

The composition of heterogenous cell types and their tightly controlled spatial localization within tissues dictates health or disease. To characterize and map these cellular identities, recently developed workflows for single-cell or spatial proteomics have demonstrated unprecedented sensitivity. The key improvements relate to automated nanoliter sample preparation and use of latest generation mass spectrometry (MS)-instrumentation for reproducible proteome analysis of single mammalian cells or small tissue sections. Despite these advances, sample acquisition throughput at biologically relevant proteome depth to provide sufficient tissue representation remains challenging. To address these limitations, we have combined a dedicated nano-96-well chip to in-line sample clean-up and chromatographic separation. In conjunction with highly sensitive and robust MS acquisition, the workflow enables robust and highly sensitive low-input proteomics, including single cell and spatial tissue analysis. Using 40SPD on the Evosep with optimized low-input dia-PASEF methods on the timsTOF Ultra2 we identify and quantify more than 4,000 proteins per single cell in solution or 25 fresh-frozen cells. To preserve the spatial context of cells within tissues we additionally combine our proteoCHIP workflow with laser capture microdissection and excise regions of interest down to 25 FFPE or fresh-frozen cells. The combination of acquisition throughput (40SPD) with this surprising proteome depth enables us to build a spatially resolved tissue catalogue including diverse tumor types and normal adjacent tissue. Based on this integrative single cell and spatial workflow we will demonstrate the analysis of clinically relevant samples.