Light-Activatable, Cell-Type Specific Labeling of the Nascent Proteome

Elucidating the mechanisms by which protein synthesis contributes to complex biological processes has remained a challenging endeavor. This is particularly true in the field of neuroscience, where multiple, tightly regulated periods of new protein synthesis in different cell-types are thought to facilitate intricate neurological functions, such as memory formation. Current methods for labeling the <i>de novo</i> proteome have lacked the spatial and temporal resolution to accurately discriminate these overlapping and often competing windows of mRNA translation. To address this technological limitation, here we describe a novel, light-inducible specific method for labeling newly synthesized proteins within a targeted cell-type.By developing Opto-ANL, a photocaged version of the nonendogenous amino acid azidonorleucine (ANL), we can selectively label newly synthesized proteins in specific cell-types through the targeted expression of a mutant methionyl-tRNA synthetase (L274G-MetRS). We demonstrate that Opto-ANL can be rapidly uncaged by UV light treatment in both cell culture and mouse brain slices, with Opto-ANL labeled proteins being able to be visualized via fluorescent noncanonical amino acid tagging. We also reveal that pretreatment with Opto-ANL not only allows for the period of <i>de novo</i> proteomic labeling to be tightly controlled, but also improves labeling efficiency compared to regular ANL. To demonstrate the potential applications of this novel technique, we use Opto-ANL to detect insulin-induced increases in protein synthesis and to label the excitatory neuronal <i>de novo</i> proteome in mouse brain slices. We believe that this application of photopharmacology will allow researchers to generate novel insights into how the translational landscape is altered across cell-types during complex neurological phenomena such as memory formation.