PXD061013-1

PXD061013 is an original dataset announced via ProteomeXchange.

Title	An optimized SP3 sample processing workflow for in-depth phos-phoproteomics and improved identification of multiply phosphory-lated peptides
Description	Protein phosphorylation is a critical and ubiquitous post-translational modification (PTM) found across the kingdoms of life. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample prepara-tion methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological capabil-ities. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have revolutionized proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C18 SPE clean-up, we demon-strate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increas-ing the number nearly 2-fold compared to standard SP3 techniques. We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently underexplored, and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.
HostingRepository	PRIDE
AnnounceDate	2025-08-25
AnnouncementXML	Submission_2025-08-24_21:51:13.124.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD061013
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	leonard daly
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-02-20 10:57:13	ID requested
⏵ 1	2025-08-24 21:51:13	announced

10.6019/PXD061013;

Daly LA, Clarke CJ, Oswald SO, Jankevics A, Brownridge PJ, Scheltema RA, Eyers CE, An Optimized SP3 Sample Processing Workflow for In-Depth and Reproducible Phosphoproteomics. J Proteome Res, 24(8):4300-4308(2025) [pubmed]

10.1021/acs.jproteome.5c00220;

submitter keyword: phosphorylation

multiply phosphorylated

mass spectrometry

PTM

SP3

TiO2

C18.

Prof Claire Eyers
contact affiliation	Centre for Proteome Research, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, U.K
contact email	ceyers@liverpool.ac.uk
lab head
leonard daly
contact affiliation	University of Liverpool
contact email	leonard.daly@liverpool.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD061013
     1. Label: PRIDE project
     2. Name: An optimized SP3 sample processing workflow for in-depth phos-phoproteomics and improved identification of multiply phosphory-lated peptides