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PXD026421

PXD026421 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleEnhanced Access to the Human Phosphoproteome with Genetically Encoded Phosphothreonine
DescriptionProtein 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.
HostingRepositoryPRIDE
AnnounceDate2022-11-11
AnnouncementXMLSubmission_2022-11-11_09:42:20.107.xml
DigitalObjectIdentifierhttps://dx.doi.org/10.6019/PXD026421
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportSupported dataset by repository
PrimarySubmitterJackMoen
SpeciesList scientific name: Escherichia coli; NCBI TaxID: 562;
ModificationListphosphorylated residue; iodoacetamide derivatized residue
InstrumentQ Exactive
Dataset History
RevisionDatetimeStatusChangeLog Entry
02021-06-02 14:40:40ID requested
12022-11-11 09:42:20announced
Publication List
10.6019/PXD026421;
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 affiliationDepartment of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA. Systems Biology Institute, Yale University, New Haven, CT 06516, USA
contact emailjesse.rinehart@yale.edu
lab head
JackMoen
contact affiliationYale University
contact emailjack.moen@yale.edu
dataset submitter
Full Dataset Link List
Dataset FTP location
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PRIDE project URI
Repository Record List
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