PXD013453-2

PXD013453 is an original dataset announced via ProteomeXchange.

Title	R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies
Description	Recent developments in mass spectrometry-based proteomics have enabled systems-level studies of cellular signaling, where >10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges and fully exploit the potential of phosphoproteomics, we introduce rapid-robotic-phosphoproteomics (R2-P2), an end-to-end automated method that processes samples in a 96-well format, from a protein extract to mass spectrometry-ready phosphopeptides. R2-P2 uses magnetic particles for both protein sample cleanup and phosphopeptide enrichment. R2-P2 is more flexible, high-throughput, rapid, and robust than classical protocols. To showcase the method, we have applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in the yeast model Saccharomyces cerevisiae. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method greatly facilitates large-scale signaling studies involving hundreds of perturbations and will open the door to systems-level studies aiming to capture the complexity of signaling.
HostingRepository	PRIDE
AnnounceDate	2019-12-12
AnnouncementXML	Submission_2022-01-07_05:19:08.087.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Mario Leutert
SpeciesList	scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	phosphorylated residue
Instrument	Orbitrap Fusion Lumos; Q Exactive; Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2019-04-11 00:05:31	ID requested
1	2019-12-12 07:54:00	announced
⏵ 2	2022-01-07 05:19:08	announced	2022-01-07: Updated publication reference for DOI(s): 1015252/msb.20199021.
3	2023-11-14 07:43:33	announced	2023-11-14: Updated project metadata.

1015252/msb.20199021;

curator keyword: Biological

submitter keyword: phosphoproteomics / signaling / MAPK / mass spectrometry / SP3 / DIA/ R2-P2

Judit Villén
contact affiliation	Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
contact email	jvillen@uw.edu
lab head
Mario Leutert
contact affiliation	Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
contact email	marioleutert@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD013453
     1. Label: PRIDE project
     2. Name: R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies