Bioorthogonal Photocatalytic Profiling of Mitochondrial Proteomes from Primary Living Samples

In situ profiling of subcellular proteomic networks in primary and living systems, such as primary cells derived from native tissues or clinic samples, is crucial for the understanding of life processes and diseases, yet challenging for the current proximity labeling methods (e.g., BioID, APEX) due to their necessity of genetic engineering. Here we report CAT-S, a new-generation bioorthogonal photocatalytic chemistry-enabled proximity labeling method, that extends proximity labeling to a wide range of primary living samples for in situ profiling of subcellular proteomes. Powered by the newly introduced thioQM labeling warhead and targeted bioorthogonal photocatalytic decaging chemistry, CAT-S enables in situ labeling of mitochondrial proteins in living cells with high efficiency and specificity. We applied CAT-S to distinct cell cultures, mouse tissues (kidney and spleen) as well as primary T cells from human blood, portraying the mitochondrial proteomic characteristics for various primary living samples, and unveiled a set of hidden mitochondrial proteins in human proteome. Furthermore, CAT-S allows quantitative analysis of the in situ proteomic perturbations on dysfunctional tissue samples, exampled by the comparative proteomics of diabetic mouse kidneys, and revealed the alterations of lipid metabolism machinery that drive the disease progression. Given the unique advantages of non-genetic operation, generality, efficiency as well as high spatiotemporal resolution, we envision that CAT-S may open new avenues as a proximity labeling strategy for in situ investigation of the native-state subcellular proteomic landscape of primary living samples that are otherwise inaccessible, promoting our understanding of the molecular mechanisms underlying biological and pathological processes.