PXD026428 is an
original dataset announced via ProteomeXchange.
Dataset Summary
Title | Enhanced Access to the Human Phosphoproteome with Genetically Encoded Phosphothreonine |
Description | Protein phosphorylation is a ubiquitous post-translational modification that governs signaling cascades and protein-protein interactions. Orthogonal translation systems repurpose evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs for the co-translational insertion of a modified amino acid. Subsequent advancements over the last decade have enabled the insertion of phospho-amino acids, bypassing a priori knowledge requirements of upstream kinases for the study of phosphoproteins. Here we optimized a pThrOTS and corresponding E. coli strain for pThr protein production. We then produced a peptidome library containing ~57,000 known threonine/phosphothreonine phosphosites using oligonucleotide library synthesis. We were able to identify approximately ~20% of the peptides encoded by the pThr library, and ~44% of the peptides encoded by the Thr library with mass spectrometry. Robust, genetically encoded phosphothreonine revealed a new activation and inhibition mechanism for the kinase CHK2. Proteome-wide surveys of interactions between active CHK2 and our peptidome library identified novel substrates and motif elements. Finally, we developed a novel technique, Hi-P+, for directly linking kinase substrate discovery to phospho-binding domain recognition, unveiling multi-level interaction networks with phosphosite resolution. This new methodology enables kinase-specific, proteome-wide surveys of multiple phosphorylation-dependent protein-protein interactions. |
HostingRepository | PRIDE |
AnnounceDate | 2022-11-11 |
AnnouncementXML | Submission_2022-11-11_09:38:31.167.xml |
DigitalObjectIdentifier | https://dx.doi.org/10.6019/PXD026428 |
ReviewLevel | Peer-reviewed dataset |
DatasetOrigin | Original dataset |
RepositorySupport | Supported dataset by repository |
PrimarySubmitter | JackMoen |
SpeciesList | scientific name: Escherichia coli; NCBI TaxID: 562; |
ModificationList | phosphorylated residue; deamidated residue; iodoacetamide derivatized residue |
Instrument | Q Exactive |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
0 | 2021-06-02 14:45:43 | ID requested | |
⏵ 1 | 2022-11-11 09:38:31 | announced | |
Publication List
Keyword List
submitter keyword: synthetic biology, proteomics, phosphoproteome, phosphoprotein interactions, Checkpoint kinase 2, 14-3-3, kinase, orthogonal translation system, genetic code expansion phosphothreonine |
Contact List
JesseRinehart |
contact affiliation | Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA. Systems Biology Institute, Yale University, New Haven, CT 06516, USA |
contact email | jesse.rinehart@yale.edu |
lab head | |
JackMoen |
contact affiliation | Yale University |
contact email | jack.moen@yale.edu |
dataset submitter | |
Full Dataset Link List
Dataset FTP location
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PRIDE project URI |
Repository Record List
[ + ]
[ - ]
- PRIDE
- PXD026428
- Label: PRIDE project
- Name: Enhanced Access to the Human Phosphoproteome with Genetically Encoded Phosphothreonine