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<p>Cell-selective bioorthogonal noncanonical amino acid tagging (BONCAT) enables time-resolved characterization of newly synthesized proteins using engineered aminoacyl-tRNA synthetases. In bacteria, this is typically accomplished using an engineered methionyl-tRNA synthetase (MetRS-NLL). Here we substantially expand the scope of this technology by introducing an engineered tyrosyl-tRNA synthetase (EcTyrRS) and a tryptophanyl-tRNA synthetase (EcTrpRS). These enzymes enable the ultrafast proteome tagging at much lower expression levels than MetRS-NLL, thereby improving both time resolution and robustness in nonmodel bacteria. Additionally, both enzymes can incorporate multiple different noncanonical amino acids with distinct click handles. This enabled new multiplexing capabilities such as distinct tagging of the nascent proteome generated in a cell in response to different cues, and tighter temporal control through pulse-chase BONCAT. EcTyrRS and EcTrpRS are also mutually orthogonal, enabling distinct proteome tagging of different cell types in mixed populations. We therefore demonstrate the utility of this technology in <i>Escherichia</i> <i>coli</i> and nonmodel ESKAPE pathogens.</p><p></p>

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Cell-selective multiplexed bioorthogonal noncanonical amino acid tagging for nascent proteomics

  • 期刊论文

  • 发表日期：

    2025年10月2日

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  • Conor Loynd,
  • Soumya Jyoti Singha Roy,
  • Sarah E. Canarelli,
  • Daniel W. Bak,
  • Bharathi Sundaresh,
  • Zachary Babbitz,
  • Tim van Opijnen,
  • Eranthie Weerapana,
  • Abhishek Chatterjee

摘要

Cell-selective bioorthogonal noncanonical amino acid tagging (BONCAT) enables time-resolved characterization of newly synthesized proteins using engineered aminoacyl-tRNA synthetases. In bacteria, this is typically accomplished using an engineered methionyl-tRNA synthetase (MetRS-NLL). Here we substantially expand the scope of this technology by introducing an engineered tyrosyl-tRNA synthetase (EcTyrRS) and a tryptophanyl-tRNA synthetase (EcTrpRS). These enzymes enable the ultrafast proteome tagging at much lower expression levels than MetRS-NLL, thereby improving both time resolution and robustness in nonmodel bacteria. Additionally, both enzymes can incorporate multiple different noncanonical amino acids with distinct click handles. This enabled new multiplexing capabilities such as distinct tagging of the nascent proteome generated in a cell in response to different cues, and tighter temporal control through pulse-chase BONCAT. EcTyrRS and EcTrpRS are also mutually orthogonal, enabling distinct proteome tagging of different cell types in mixed populations. We therefore demonstrate the utility of this technology in Escherichia coli and nonmodel ESKAPE pathogens.