Bioorthogonal Photocatalytic Decaging-Enabled Mitochondrial Proteomics

Abstract

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.

Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

“…Notably, this protein inventory has exceptionally high mitochondrial specificity, with 97% (383 out of 394) of proteins listed in the MitoCarta 3.0, which is higher than previously reported proximity labeling methods, including APEX (92%) 5 , TurboID (60%) 8 , and small molecule photosensitizer-based CAT-Prox (70%) 22 (Figure 3C). In terms of sub-mitochondrial specificity, 64% and 30% of our RinID dataset are mitochondrial matrix and inner membrane proteins, respectively (Figure 3D).…”

Seeking Citius: Photochemical Access of Reactive Intermediates for Faster Bioorthogonal Reactions

“…To this end, some prominent photochemical precursors include tetrazoles, [7] sydnones, [8] azirines, [9] and quinones [10] . Besides bioorthogonal labeling, photogenerated reactive intermediates such as quinone methides also permit the profiling of protein−protein and protein−DNA complexes [11] as well as mitochondrial proteomes in living cells [12] . Herein, we focus our discussion on recent advances in tetrazole photoclick chemistry and photo‐triggered tetrazine ligation because of their superior reaction kinetics and greater compatibility with biological systems.…”

Photochemical Identification of Auxiliary Severe Acute Respiratory Syndrome Coronavirus 2 Host Entry Factors Using μMap

“… 20 This platform has been applied for small molecule target identification, 21 mapping chromatin reorganization events, 22 and rapidly profiling immunosynapse interactions. 20 Additionally, similar Ir-based photocatalytic proximity labeling strategies have also been recently developed for cataloging mitochondrial proteins in activated macrophages 23 and profiling surface proteins in breast cancer. 24 Rhodamine-based oxidation labeling has also provided new methodology for validating cell–surface microenvironments, underscoring the utility of light-mediated proximity labeling for interrogating cell–surface proteomes with high spatiotemporal precision.…”