In-depth and high-throughput spatial proteomics for whole-tissue slice profiling by deep learning-facilitated sparse sampling strategy

Mammalian organs and tissues are composed of heterogeneously distributed cells, which interact with each other and the extracellular matrix surrounding them in a spatially defined way. Therefore, spatially resolved gene expression profiling is crucial for determining the function and phenotypes of these cells. While genome mutations and transcriptome alterations act as drivers of diseases, the proteins that they encode regulate essentially all biological functions and constitute the majority of biomarkers and drug targets for disease diagnostics and treatment. However, unlike transcriptomics, which has a recent explosion in high-throughput spatial technologies with deep coverage, spatial proteomics capable of reaching bulk tissue-level coverage is still rare in the field, due to the non-amplifiable nature of proteins and sensitivity limitation of mass spectrometry (MS). More importantly, due to the limited multiplexing capability of the current proteomics methods, whole-tissue slice mapping with high spatial resolution requires a formidable amount of MS matching time. To achieve spatially resolved, deeply covered proteome mapping for centimeter-sized samples, we developed a sparse sampling strategy for spatial proteomics (S4P) using computationally assisted image reconstruction methods, which is potentially capable of reducing the number of samples by tens to thousands of times depending on the spatial resolution. In this way, we generated the largest spatial proteome to date, mapping more than 9000 proteins in the mouse brain, and discovered potential new regional or cell type markers. Considering its advantage in sensitivity and throughput, we expect that the S4P strategy will be applicable to a wide range of tissues in future studies.