Dual‐Responsive Nanogel for Mitochondria‐Targeted Delivery and Release of Cross‐Linkers to Decipher In Vivo Protein Conformations and Interactions

ABSTRACT Mitochondrial proteins play essential roles in energy metabolism, transmembrane transport, and membrane structural maintenance. The deep coverage analysis of in situ protein conformations and interactions in the mitochondria of living cells remains challenging, due to the limited delivery and release efficiency of cross‐linkers. Herein, we developed a mitochondria‐targeted and dual‐responsive nanogel system that integrated reactive oxygen species (ROS)‐cleavable thioketal bonds and thermosensitive phase‐transition elements for mitochondria‐specific delivery and fast release of cross‐linkers. Compared to previous polymeric micelle systems, the nanogel showed expansive compatibility with hydrophilic cross‐linkers, whose efficient release under the elevated ROS and temperature of mitochondria contributed to high cross‐linking coverage. Using this nanogel system, 3927 intra‐links and 904 inter‐links across 783 mitochondrial proteins were identified, representing a 2.3‐fold increase in the number of cross‐links compared to the previously developed ROS‐responsive polymeric micelle system. These data revealed the possible existence of ATP synthasome and flexible or membrane‐associated conformations inaccessible to crystallography. This work establishes an efficient strategy for in situ conformation and interaction analysis of mitochondrial proteins in the native environment, which is helpful for understanding the mitochondrial protein function.