PXD002344-2

PXD002344 is an original dataset announced via ProteomeXchange.

Title	Global and phosphoproteomic profiling of the DNA damage response in yeast by SILAC labeling and LC-MS/MS
Description	Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.
HostingRepository	PRIDE
AnnounceDate	2016-04-04
AnnouncementXML	Submission_2016-04-06_00:23:13.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Jacob Kennedy
SpeciesList	scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	phosphorylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2015-06-10 02:54:04	ID requested
1	2016-04-04 06:21:20	announced
⏵ 2	2016-04-06 00:23:15	announced	Updated publication reference for PubMed record(s): 27017623.

Huang D, Piening BD, Kennedy JJ, Lin C, Jones-Weinert CW, Yan P, Paulovich AG, DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae. Genetics, 203(1):353-68(2016) [pubmed]

curator keyword: Biological

submitter keyword: Methylmethanesulfonate, Mass spectrometry, Phosphorylation, DNA damage checkpoint, Genetic interaction, Homologous recombination, telomere, DNA damage response

Amanda G Paulovich
contact affiliation	Fred Hutchinson Cancer Research Center
contact email	apaulovi@fredhutch.org
lab head
Jacob Kennedy
contact affiliation	Clinical Research Division
contact email	jkennedy@fhcrc.org
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/2016/04/PXD002344

PRIDE project URI

• PRIDE
  1. PXD002344
     1. Label: PRIDE project
     2. Name: Global and phosphoproteomic profiling of the DNA damage response in yeast by SILAC labeling and LC-MS/MS