title: Tagging and Enriching Proteins Enables Cell-Specific Proteomics.
abstract: Cell-specific proteomics in multicellular systems and whole animals is a promising approach to understand the differentiated functions of cells and tissues. Here, we extend our stochastic orthogonal recoding of translation (SORT) approach for the co-translational tagging of proteomes with a cyclopropene-containing amino acid in response to diverse codons in genetically targeted cells, and create a tetrazine-biotin probe containing a cleavable linker that offers a way to enrich and identify tagged proteins. We demonstrate that SORT with enrichment, SORT-E, efficiently recovers and enriches SORT tagged proteins and enables specific identification of enriched proteins via mass spectrometry, including low-abundance proteins. We show that tagging at distinct codons enriches overlapping, but distinct sets of proteins, suggesting that tagging at more than one codon enhances proteome coverage. Using SORT-E, we accomplish cell-specific proteomics in the fly. These results suggest that SORT-E will enable the definition of cell-specific proteomes in animals during development, disease progression, and learning and memory.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Elliott
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Ambra
authors.initials: A
authors.lastname: Bianco
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Townsley
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Fried
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address: chin@mrc-lmb.cam.ac.uk.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001391:Azo Compounds / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology*; D005260:Female; D005815:Genetic Code; D013058:Mass Spectrometry; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry*; D015394:Molecular Structure; D021381:Protein Transport; D040901:Proteomics; D013777:Tetrazoles / Q000737:chemistry
chemical_list: D000596:Amino Acids; D001391:Azo Compounds; D029721:Drosophila Proteins; D015335:Molecular Probes; D013777:Tetrazoles; D001710:Biotin; C009850:azobenzene