A Set of Organelle-Localizable Reactive Molecules for Mitochondrial Chemical Proteomics in Living Cells and Brain Tissues

Protein functions
are tightly regulated by their subcellular localization
in live cells, and quantitative evaluation of dynamically altered
proteomes in each organelle should provide valuable information. Here,
we describe a novel method for organelle-focused chemical proteomics
using spatially limited reactions. In this work, mitochondria-localizable
reactive molecules (MRMs) were designed that penetrate biomembranes
and spontaneously concentrate in mitochondria, where protein labeling
is facilitated by the condensation effect. The combination of this
selective labeling and liquid chromatography–mass spectrometry
(LC–MS) based proteomics technology facilitated identification
of mitochondrial proteomes and the profile of the intrinsic reactivity
of amino acids tethered to proteins expressed in live cultured cells,
primary neurons and brain slices. Furthermore, quantitative profiling
of mitochondrial proteins whose expression levels change significantly
during an oxidant-induced apoptotic process was performed by combination
of this MRMs-based method with a standard quantitative MS technique
(SILAC: stable isotope labeling by amino acids in cell culture). The
use of a set of MRMs represents a powerful tool for chemical proteomics
to elucidate mitochondria-associated biological events and diseases.