Spatiotemporally
resolved dissection of subcellular proteome is
crucial to our understanding of cellular functions in health and disease.
We herein report a bioorthogonal and photocatalytic decaging-enabled
proximity labeling strategy (CAT-Prox) for spatiotemporally resolved
mitochondrial proteome profiling in living cells. Our systematic survey
of the photocatalysts has led to the identification of Ir(ppy)2bpy as a bioorthogonal and mitochondria-targeting catalyst
that allowed photocontrolled, rapid rescue of azidobenzyl-caged quinone
methide as a highly reactive Michael acceptor for proximity-based
protein labeling in mitochondria of live cells. Upon careful validation
through in vitro labeling, mitochondria-targeting
specificity, in situ catalytic activity as well as
protein tagging, we applied CAT-Prox for mitochondria proteome profiling
in living Hela cells as well as hard-to-transfect macrophage RAW264.7
cells with approximately 70% mitochondria specificity observed from
up to 300 proteins enriched. Finally, CAT-Prox was further applied
to the dynamic dissection of mitochondria proteome of macrophage cells
upon lipopolysaccharide stimulation. By integrating photocatalytic
decaging chemistry with proximity-based protein labeling, CAT-Prox
offers a general, catalytic, and nongenetic alternative to the enzyme-based
proximity labeling strategies for diverse live cell settings.