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J Am Chem Soc. 2018 Jun 13;140(23):7046-7051. doi: 10.1021/jacs.8b03074. Epub 2018 May 30.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

Author information

1
Center for Tissue Regeneration, Repair and Restoration , V.A. Palo Alto Healthcare System , Palo Alto , California 94304 , United States.

Abstract

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

PMID:
29775058
PMCID:
PMC6598694
DOI:
10.1021/jacs.8b03074
[Indexed for MEDLINE]
Free PMC Article

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