Near-Infrared Light-Mediated Living Cells Mitochondrial Proteome 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.