Cell type-specific proteomic profiling in Caenorhabditis elegans

Abstract

In a complex eukaryote like C. Elegans, cell and tissue heterogeneity restricts the usefulness of large-scale, mass spectrometrybased anal. of an organism's proteome. As worm cell lines are not available and enriching for specific cells or tissues is challenging, researchers cannot systematically identify low abundance proteins expressed in specific cells or tissues from wholeworm lysates. To isolate protein from specific cells, we have engineered C. Elegans phenylalanyl-tRNA synthetases capable of appending azide- or alkynyl-bearing analogs of phenylalanine to tRNA^(Phe). As these analogs are not substrates for any of the wild-type aminoacyl-tRNA synthetases in C. Elegans, we achieved cell type selectivity by spatially restricting the expression of a mutant synthetase using cell type-specific promoters. Because proteins from those cells that express the mutant synthetase - and only those cells - contain the analogs, they can be enriched by treatment with alkyne- or azide-functionalized biotin reagents and subsequent affinity chromatog. for identification by LC/MS-MS.