Cell-selective labelling of proteomes in Drosophila melanogaster

The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRS LtoG ) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through ‘click chemistry’. To test these methods for applicability in vivo , we expressed MetRS LtoG cell specifically in Drosophila . FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms. Mutated tRNA synthetases can incorporate non-canonical amino acids into proteins. Erdmann et al. exploit this property to metabolically label newly synthesized proteins in selected cell types in Drosophila , and demonstrate their detection using proteomics (BONCAT) and fluorescence imaging (FUNCAT).