Updated project metadata. Puromycin and its derivative O-propargyl puromycin (OPP) enable detection of 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 have used puromycin N-acetyltransferase (PAT), for cell-specific metabolic labelling with puromycin or OPP. This approach, which we named Puromycin Inactivation for Cell-Selective proteome Labelling (PICSL), is based on efficient inactivation of puromycin or OPP in cells expressing PAT and detection of translation in other cell types. We performed two mass spectrometry experiments using cell-specific expression of PAT, metabolic labeling of cells with OPP and streptavidin pulldown of biotinylated de novo synthesized proteins. In the first proof-of-principle experiment (part I) we mixed human (HEK293) and mouse (Neuro2a) cells and labeled them with OPP, comparing this with another sample where HEK293 cells expressed PAT. We show specific reduction of human-specific peptides in de novo synthesized proteome in HEK-PAT cells. In the second experiment (part II), we used a primary co-culture of rat cortical neurons (expressing PAT in some samples) and glia, performed OPP labeling and pulldown of de novo synthesized proteins.