Cell type-specific labeling of newly synthesized proteins by puromycin inactivation.

ABSTRACT: Puromycin and its derivative O-propargyl puromycin (OPP) have recently found widespread use in detecting nascent proteins. Use of these metabolic labels in complex mixtures of cells leads to indiscriminate tagging of nascent proteomes independent of cell type. Here, we show how a widely used mammalian selection marker, puromycin N-acetyltransferase, can be repurposed for cell-specific metabolic labeling. This approach, which we named puromycin inactivation for cell-selective proteome labeling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing puromycin N-acetyltransferase and detection of translation in other cell types. Using cocultures of neurons and glial cells from the rat brain cortex, we show the application of PICSL for puromycin immunostaining, Western blot, and mass spectrometric identification of nascent proteins. By combining PICSL and OPP-mediated proteomics, cell type-enriched proteins can be identified based on reduced OPP labeling in the cell type of interest.

SUBMITTER: Cabrera-Cabrera F 

PROVIDER: S-EPMC10497999 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature