Cell-selective multiplexed bioorthogonal noncanonical amino acid tagging for versatile nascent proteome profiling

ABSTRACT: Cell-selective bioorthogonal noncanonical amino acid tagging (BONCAT) of newly synthesized proteins, catalyzed by engineered aminoacyl-tRNA synthetase (aaRSs), is a powerful technology for time-resolved characterization of the nascent proteome. Currently, cell-selective BONCAT studies in bacteria rely largely on an engineered methionyl-tRNA synthetase (MetRS-NLL) that labels the nascent proteome with azidonorleucine. Here, we expand the scope of BONCAT in bacteria by introducing two new classes of engineered aaRSs, a tyrosyl-tRNA synthetase (EcTyrRS) and a tryptophanyl-tRNA synthetase (EcTrpRS). These aaRSs enable ultra-fast proteome tagging at much lower expression levels relative to the current gold-standard MetRS-NLL, improving both the time resolution of BONCAT, as well as the robustness of the technology in non-model bacteria. Simultaneous use of EcTyrRS+EcTrpRS enabled more efficient enrichment of the nascent proteome. Moreover, both aaRSs can accept multiple different ncAAs with distinct click handles – or no click handles – unlocking new multiplexing capabilities. For instance, newly synthesized proteins generated in response to two different cues in the same cell can be distinctly tagged with two different click-ncAAs for their separate characterization. It also allows temporal control over BONCAT, where labeling is initiated with a ‘pulse’ of a click-ncAA followed by its quenching with a ‘chase’ of a non-click ncAA. Furthermore, EcTyrRS and EcTrpRS are mutually orthogonal, enabling distinct proteome tagging of two different cell-types in a mixed population. Finally, we demonstrate the utility of these tools in E. coli, as well as in non-model ESKAPE pathogens Klebsiella pneumoniae and Acinetobacter baumannii.

INSTRUMENT(S):

ORGANISM(S): Acinetobacter Baumannii (strain Atcc 17978 / Cip 53.77 / Lmg 1025 / Ncdc Kc755 / 5377) Klebsiella Pneumoniae Atcc 43816 Escherichia Coli

SUBMITTER: Daniel Bak  

LAB HEAD: Eranthie Weerapana

PROVIDER: PXD067013 | Pride | 2026-04-11

REPOSITORIES: Pride