Near-InfraredLight-Mediated Living Cells MitochondrialProteome Proximity Labeling

Photocatalytic proximity labeling techniques enable significant advances in understanding the subcellular proteome. However, current methods primarily utilize visible light that suffers from low labeling efficiency and limited identification coverage due to absorption overlap with endogenous chromophores and scattering in biological samples. To address these issues, we reported the proximity labeling strategy using near-infrared excitation (PL-NIR) strategy, a proximity labeling platform that used a near-infrared-excited catalyst to activate labeling mediated by reactive oxygen species, which could avoid the disadvantages of visible light. Taking advantage of the near-infrared excitation and mitochondrial targeting properties of IR780, the mitochondrial proteome were selectively labeled by spatially limited reaction in the native environment. The PL-NIR strategy facilitated the plotting of the mitochondrial proteome in which up to 245 mitochondrial proteins were identified in living HeLa cells. Compared with the current methods, the PL-NIR strategy significantly increased identification coverage, especially for mitochondrial inner membrane proteins. Furthermore, mitochondrial proteome dynamics were deciphered in LPS stimulated HMC3 cells, which were hard to transfect. Overall, the PL-NIR strategy as a highly precise proteomic platform offered improved identification coverage for more knowledge of subcellular biology discovering.