I have developed a far-red light mediated proximal labeling (LIMPLA) strategy for subcellular protein profiling in live cells. This strategy can facilitate the proteomic profiling of subcellular organelle, such as the mitochondria with no need of transgene. I modified the mitochondria-targeting dye rhodamine 123 with an alkynyl group (Alk-R123). These modifications do not change the mitochondria targeting property and can act as a bioorthogonal 'handle' for subsequently imaging or enrichment. After Alk-R123 are accumulated in the mitochondria, light illumination can activate the dye to label proximal proteins in situ. In order to enhance protein labeling efficiency, I have found that various mitochondria-targeting dyes such as Mitotracker deep red can mediate 2-Propynylamine (PA) to label proximal proteins under illumination. PA tagged proteins with alkynyl group are subsequently derivatized via click chemistry with azido fluorescent dye for imaging or with azido biotin for further enrichment and mass-spec identification.
Main textEukaryotic cells are elaborately subdivided into functionally distinct, membraneenclosed compartments or organelles. Each organelle contains its own characteristic set of proteins, which underlie its characteristic structural and functional properties. For example, mitochondria, a double-membrane-bound organelle, are energy center and plays crucial roles in energy conversion and calcium ions storage. Understanding how organelles function appropriately and specifically requires identifications of proteomics in specific organelles.