PXD062515

PXD062515 is an original dataset announced via ProteomeXchange.

Title	Scalable Phosphotyrosine Enrichment with SH2 Superbinder Enables Deep Profiling of EGF Responses
Description	Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, proteomic studies of tyrosine phosphorylation have been limited in scale and throughput by costly reagents and labor-intensive protocols. To address this, we developed R2HaPpY, a phosphotyrosine enrichment method combining simplified superbinder reagent preparation and automated high-throughput enrichment. Our new reagent binds phosphotyrosine peptides at higher efficiency than existing enrichment reagents and reduces both cost and preparation time by 20-fold. We generalized R2HaPpY to samples of low and high phosphotyrosine levels, and benchmarked it on EGF signaling dynamics in HeLa cells. Using only ~1 mg of input peptides, we quantify 1,651 unique phosphotyrosine sites, including 878 regulated phosphotyrosine sites, many novel or not previously annotated as EGF-responsive. Our results reveal differential temporal regulation and represent the largest phosphotyrosine dataset of EGF stimulation response to date. This streamlined, cost-effective, and sensitive method enables quantitative mapping of tyrosine phosphorylation dynamics at the scale of hundreds of samples, facilitating integration of phosphotyrosine signaling into multiomic studies across diverse biological systems.
HostingRepository	PRIDE
AnnounceDate	2026-07-03
AnnouncementXML	Submission_2026-07-03_13:23:36.737.xml
DigitalObjectIdentifier	https://doi.org/10.6019/PXD062515
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Alexis Chang
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606; scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: NEWT:4932; scientific name: Saccharomyces cerevisiae; NCBI TaxID: NCBITaxon:4932;
ModificationList	phosphorylated residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2025-04-02 04:14:36	ID requested
⏵ 1	2026-07-03 13:23:37	announced

10.1038/S44318-026-00843-8;

10.6019/PXD062515;

submitter keyword: phosphotyrosine, automated, HeLa, enrichment, high-throughput, EGF, LC-MS/MS

Judit Villen
contact affiliation	Department of Genome Sciences; Villen Lab; University of Washington; United States
contact email	jvillen@uw.edu
lab head
Alexis Chang
contact affiliation	University of Washington
contact email	alexisn1@uw.edu
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/2026/07/PXD062515

PRIDE project URI

• PRIDE
  1. PXD062515
     1. Label: PRIDE project
     2. Name: Scalable Phosphotyrosine Enrichment with SH2 Superbinder Enables Deep Profiling of EGF Responses