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PXD014525

PXD014525 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleRapid and site-specific deep phosphoproteome profiling by data-independent acquisition (DIA) without the need for spectral libraries
DescriptionQuantitative phosphoproteomics has in recent years revolutionized understanding of cell signaling, but it remains a challenge to scale the technology for high-throughput analyses. Here we present a rapid and reproducible phosphoproteomics approach to systematically analyze hundreds of samples by fast liquid chromatography tandem mass spectrometry using data independent acquisition (DIA). To overcome the inherent issue of positional phosphopeptide isomers in DIA-based phosphoproteomics, we developed and employed an accurate site localization scoring algorithm, which is incorporated into the Spectronaut software tool. Using a library of synthetic phosphopeptides spiked-in to a yeast phosphoproteome in different ratios we show that it is on par with the top site localization score for data-dependent acquisition (DDA) based phosphoproteomics. Single-shot DIA-based phosphoproteomics achieved an order of magnitude broader dynamic range, higher reproducibility of identification and improved sensitivity and accuracy of quantification compared to state-of-the-art DDA-based phosphoproteomics. Importantly, direct DIA without the need of spectral libraries performed almost on par with analyses using specific project-specific libraries. Moreover, we implemented and benchmarked an algorithm for globally determining phosphorylation site stoichiometry in DIA. Finally, we demonstrate the scalability of the DIA approach by systematically analyzing the effects of thirty different kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that a large proportion of EGF-dependent phospho-regulation is mediated by a specific set of protein kinases.
HostingRepositoryPRIDE
AnnounceDate2020-02-18
AnnouncementXMLSubmission_2020-02-18_10:01:28.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterDorte Bekker-Jensen
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListphosphorylated residue
InstrumentQ Exactive
Dataset History
RevisionDatetimeStatusChangeLog Entry
02019-07-09 01:50:39ID requested
12020-02-18 10:01:29announced
Publication List
Bekker-Jensen DB, Bernhardt OM, Hogrebe A, Martinez-Val A, Verbeke L, Gandhi T, Kelstrup CD, Reiter L, Olsen JV, Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition without the need for spectral libraries. Nat Commun, 11(1):787(2020) [pubmed]
Keyword List
submitter keyword: DIA, Phosphorylation, Q Exactive HF-X, Rapid, Sensitive
Contact List
Jesper V. Olsen
contact affiliationThe Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark
contact emailjesper.olsen@cpr.ku.dk
lab head
Dorte Bekker-Jensen
contact affiliationUniversity of Copenhagen
contact emaildorte.bekker-jensen@cpr.ku.dk
dataset submitter
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PRIDE project URI
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