PXD067013-1

PXD067013 is an original dataset announced via ProteomeXchange.

Title	Cell-selective multiplexed bioorthogonal noncanonical amino acid tagging for versatile nascent proteome profiling
Description	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.
HostingRepository	PRIDE
AnnounceDate	2026-04-11
AnnouncementXML	Submission_2026-04-11_10:46:42.828.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Daniel Bak
SpeciesList	scientific name: Klebsiella pneumoniae ATCC 43816; NCBI TaxID: NEWT:1316582; scientific name: Escherichia coli; NCBI TaxID: NEWT:562; scientific name: Acinetobacter baumannii (strain ATCC 17978 / CIP 53.77 / LMG 1025 / NCDC KC755 / 5377); NCBI TaxID: NEWT:400667;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue; dimethylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 240; LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2025-08-06 14:15:58	ID requested
⏵ 1	2026-04-11 10:46:43	announced

10.1038/S41589-025-02039-3;

submitter keyword: Chemoproteomics, BONCAT, Nascent Proteins

Eranthie Weerapana
contact affiliation	Chemistry Department, Boston College
contact email	eranthie@bc.edu
lab head
Daniel Bak
contact affiliation	Boston College
contact email	bakd@bc.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD067013

PRIDE project URI

• PRIDE
  1. PXD067013
     1. Label: PRIDE project
     2. Name: Cell-selective multiplexed bioorthogonal noncanonical amino acid tagging for versatile nascent proteome profiling