PXD014525-1

PXD014525 is an original dataset announced via ProteomeXchange.

Title	Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition (DIA) without the need for spectral libraries
Description	Quantitative 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.
HostingRepository	PRIDE
AnnounceDate	2020-02-18
AnnouncementXML	Submission_2020-02-18_10:01:28.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dorte Bekker-Jensen
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2019-07-09 01:50:39	ID requested
⏵ 1	2020-02-18 10:01:29	announced
2	2024-10-22 05:04:11	announced	2024-10-22: Updated project metadata.

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]

submitter keyword: DIA, Phosphorylation, Q Exactive HF-X, Rapid, Sensitive

Jesper V. Olsen
contact affiliation	The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark
contact email	jesper.olsen@cpr.ku.dk
lab head
Dorte Bekker-Jensen
contact affiliation	University of Copenhagen
contact email	dorte.bekker-jensen@cpr.ku.dk
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/2020/02/PXD014525

PRIDE project URI

• PRIDE
  1. PXD014525
     1. Label: PRIDE project
     2. Name: Rapid and site-specific deep phosphoproteome profiling by data-independent acquisition (DIA) without the need for spectral libraries