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

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.