PXD019815-2

PXD019815 is an original dataset announced via ProteomeXchange.

Title	Streamlined identification of permissive non-canonical amino acid incorporation sites in mammalian cell lines with an expanded genetic code
Description	The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at www.bultmannlab.eu/tools/iPASS), a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_05:19:11.774.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Enes Ugur
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; acetylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2020-06-16 08:31:30	ID requested
1	2021-02-23 07:14:56	announced
⏵ 2	2024-10-22 05:19:12	announced	2024-10-22: Updated project metadata.

Dataset with its publication pending

submitter keyword: Genetic code expansion, unnatural amino acids, non-canonical amino acids, amber suppression

Michael Wierer
contact affiliation	Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany
contact email	wierer@biochem.mpg.de
lab head
Enes Ugur
contact affiliation	Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry
contact email	ugur@bio.lmu.de
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD019815
     1. Label: PRIDE project
     2. Name: Streamlined identification of permissive non-canonical amino acid incorporation sites in mammalian cell lines with an expanded genetic code