Visualizing and profiling de novo protein synthesis in Drosophila with cell-type specificity

Gene expression is frequently regulated with cell type specificity and at multiple levels, including through tightly-controlled protein synthesis. While existing methods of protein synthesis profiling with non-canonical amino acid or isotope-containing amino acid labeling are effective and efficient for use in cultured cells, they are often costly to carry out in vivo. We previously developed a method to visualize and capture nascent proteomes in a cell type-specific manner in Drosophila brain explants using phenylacetyl-OPP (PhAc-OPP), a modified form of the puromycin analog O-propargyl-puromycin (OPP) that can incorporate into growing polypeptide chains. Targeted expression of Penicillin G acylase (PGA) in a cell population of interest removes an enzyme-labile blocking group on PhAc-OPP, thus permitting OPP-dependent labeling of nascent proteins in that cell population. This method, which we call POPPi (PGA-dependent OPP incorporation), provides a versatile approach to visualize or identify proteins synthesized in cell populations of interest in vivo. Here, we provide detailed protocols for labeling newly-synthesized protein in Drosophila brain cell populations by POPPi followed by detection via immunofluorescence or by capture and protein identification.