Cell type-specific proteomic profiling in Caenorhabditis elegans

Abstract

Caenorhabditis elegans is a small, free-living soil nematode. For several decades. this multi-cellular organism has been used to study intricate biological and molecular processes in fields ranging from developmental biology to neurobiology. In a complex eukaryote like C. elegans, cell and tissue heterogeneity restricts the usefulness oflarge-scale, mass spectrometrybased analysis 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 specifc cells or tissues from whole-worm lysates. To isolate protein from specific cells, we have engineered a family of mutant C. elegans phenylalanyl-tRNA synthetases capable of appending azide- or alkynyl-bearing analogs of phenylalanine to tRNAPhe. As these analogs are not substrates for any of the wild-type aminoacyltRNA 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 alkyneor azide-functionalized biotin reagents and subsequent affinity chromatography for identification by LC/MSMS.

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