PXD045601-2

PXD045601 is an original dataset announced via ProteomeXchange.

Title	Systematic evaluation of experimental parameters for sensitive phosphoproteomics analysis
Description	The field of phosphoproteomics has been revolutionized in the last years, with enhanced efficiency and sensitivity, now enabling deep phosphoproteomic analysis single-shot from minimal peptide inputs. In this study, we rigorously evaluate the effects of various experimental parameters in automated Zirconium-IMAC (Zr-IMAC) based phosphoproteomic sample preparation with focus on low peptide input samples. Assessing metrics such as number of identified phosphopeptides, selectivity, phosphosite localization scores and relative purification of multiply phosphorylated phosphopeptides, we were able to pinpoint key influencing variables. Determination of the optimal glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide to beads ratio, binding time, sample volume and loading buffer volume, allowed us to confidently localize up to ~15,800 phosphopeptides using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost the depth of the analysis, and most importantly, whether pooling fractions into a single-LCMS analysis also increases the phosphoproteome depth. As a result, we present a novel strategy based on incremental addition of beads and subsequent fraction pooling, which can boost by 17% the phosphoproteome coverage when compared to standard enrichment.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:34:41.479.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ana Martinez-Val
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2023-09-22 11:34:18	ID requested
1	2024-03-26 20:50:13	announced
⏵ 2	2024-10-22 06:34:41	announced	2024-10-22: Updated project metadata.

10.1016/J.MCPRO.2024.100754;

submitter keyword: Phosphoproteomics, Evosep, sequential enrichment

Jesper V. Olsen
contact affiliation	Vice Director, Professor University of Copenhagen Faculty of Health and Medical Sciences Novo Nordisk Foundation Center for Protein Research Proteomics Program Blegdamsvej 3B, DK-2200 Copenhagen N Denmark
contact email	jesper.olsen@cpr.ku.dk
lab head
Ana Martinez-Val
contact affiliation	Novo Nordisk Foundation Center for Protein Research
contact email	ana.mdval@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/2024/03/PXD045601

PRIDE project URI

• PRIDE
  1. PXD045601
     1. Label: PRIDE project
     2. Name: Systematic evaluation of experimental parameters for sensitive phosphoproteomics analysis