PXD031277-2

PXD031277 is an original dataset announced via ProteomeXchange.

Title	Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing
Description	In large-scale quantitative mass spectrometry (MS)-based phosphoproteomics, isobaric labeling with tandem mass tags (TMTs) coupled with offline high-pH reversed-phase peptide chromatographic fractionation maximizes depth of coverage. To investigate to what extent limited sample amounts affects sensitivity and dynamic range of the analysis due to sample losses, we benchmarked TMT-based peptide fractionation strategies against single-shot (SS) label-free approach with data independent acquisition (DIA), for different peptide input per sample. To systematically examine how peptide input amounts influence TMT-fractionation approaches in a phosphoproteomics workflow, we compared two different high-pH reverse-phase fractionation strategies, microflow (MF) and stage-tip fractionation (STF), while scaling the peptide input amount down from 12.5 μg to 1 μg per sample. Our results indicate that, for input amounts higher than 5 μg per sample, TMT labeling, followed by microflow fractionation and phospho-enrichment (MF), achieves the deepest phosphoproteome coverage, even compared to SS DIA analysis. Conversely, stage-tip fractionation of enriched phosphopeptides (STF) is optimal for lower amounts, below 5 μg/peptide per sample. As a result, we provide a decision tree to help phosphoproteomics users to choose the best workflow as a function of on sample amount.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:54:49.637.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Claire Koenig
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2022-01-26 12:35:16	ID requested
1	2022-06-28 10:30:13	announced
⏵ 2	2023-11-14 08:54:50	announced	2023-11-14: Updated project metadata.

Dataset with its publication pending

submitter keyword: Tandem mass tags,Phosphoproteomics, optimization, high-pH fractionation, Isobaric labeling

Jesper V. Olsen
contact affiliation	Group leader, Professor University of Copenhagen Faculty of Health and Medical Sciences Blegdamsvej 3B København N
contact email	jesper.olsen@cpr.ku.dk
lab head
Claire Koenig
contact affiliation	Novo Nordisk Foundation Center for Protein Research, University of Copenhagen
contact email	claire.koenig@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/2022/06/PXD031277

PRIDE project URI

• PRIDE
  1. PXD031277
     1. Label: PRIDE project
     2. Name: Optimal analytical strategies for sensitive and quantitative phosphoproteomics using TMT-based multiplexing