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.
Pubmed ID: 26138272 RIS Download
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Software application for genetic analysis of human pedigree data under models involving a small number of loci. MENDEL is useful for segregation analysis, linkage calculations, genetic counseling, allele frequency estimation, and related kinds of problems. (entry from Genetic Analysis Software)
View all literature mentionsDrosophila melanogaster with name w[1118]; P{w[+mW.hs]=GawB}VGlut[OK371] from BDSC.
View all literature mentionsDrosophila melanogaster with name w[1118]; P{w[+m*]=GAL4}repo/TM3, Sb[1] from BDSC.
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