PXD000659

PXD000659 is an original dataset announced via ProteomeXchange.

Title	The circadian clock output deubiquitylating enzyme USP2-45 regulates intestinal Ca2+ absorption
Description	In order to identify molecular targets of USP2-45 in the small intestine, we carried out a quantitative proteomics iTraq (Isobaric tag for relative and absolute quantitation) screen to compare proteins from WT and knockout mice. With this analysis we identified, among others, the scaffolding protein Na(+)/H(+) Exchange Regulatory cofactor NHERF4 to be up-regulated (4.81-fold) in the duodenum of Usp2-KO mice. Methods : Usp2-KO and WT littermates males were entrained to 12h L:12h D for at least two weeks in light-light housing boxes. Mice were sacrificed by cervical disolcation at ZT13 under red dim light and duodenal mucosa was dissected by scraping. The tissue samples were quickly frozen in liquid nitrogen before total protein extraction. Proteins were extracted in a lysis buffer containing 8M Urea, 20mM Tris-HCl, pH 7.5, 14 µg/ml Aprotinin, 0.7 µg /ml PepstatinA 0.7 µg /ml Leupeptin, 1 mM NaVO4, 10 mM Na-Pyrophosphate, 50 µM MG132 and 1mM PMSF. Protein concentration was measured by Bradford quantitation. Relative quantitation of proteins in 4 pools of 3 KO and 4 pools of 3 WT tissue samples was carried out by iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). After reduction and alkylation of cysteines, proteins were precipitated, redissolved in 8M Urea buffer and digested with trypsin (1:50 w:w) overnight. 80ug of digested material for each sample was labeled by reaction with one vial of iTRAQ 8-plex reagent (ABSciex). Samples were pooled , desalted and fractionated by off-gel electrofocusing. The 24 fractions obtained were analysed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) on a hybrid linear trap LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) on a two-hour gradient. Full MS survey scans were performed at 60’000 resolution and the ten most intense multiply charged precursor ions detected in the full MS survey scan were selected for HCD (Higher energy Collision Dissociation) fragmentation and Orbitrap analysis (7500 resolution). Each spectrum was acquired at two relative collision energies (35% and 45%) and the spectra were summed. Data files were analysed with MaxQuant 1.3.0.5 incorporating the Andromeda search engine. Further data analysis was performed using the R statistical programming language version 2.15.2 (R core team 2012). Intensities for the reporter ions 113 to 121 were normalized using the Variance Stabilizing method (R package version 3.26.0). Proteins with non-zero intensities in all channels were evaluated for differential expression using the Local-Pooled-Error method (R package version 1.32.0) followed by correction for multiple testing according to Benjamini and Hochberg.
HostingRepository	PRIDE
AnnounceDate	2016-03-02
AnnouncementXML	Submission_2016-03-02_02:19:18.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Daniel Pouly
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	iTRAQ8plex-116 reporter+balance reagent acylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-12-19 02:05:41	ID requested
⏵ 1	2016-03-02 02:19:19	announced

Pouly D, Chenaux S, Martin V, Babis M, Koch R, Nagoshi E, Katanaev VL, Gachon F, Staub O, USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level. PLoS One, 11(1):e0145155(2016) [pubmed]

submitter keyword: Intestinal calcium absorption, circadian clock, ubiquitin-specific protease 2, isobaric tagging, mass spectrometry

Olivier Staub
contact affiliation	Department of Pharmacology & Toxicology, University of Lausanne, Switzerland
contact email	staub@unil.ch
lab head
Daniel Pouly
contact affiliation	Department of Pharmacology and Toxicology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
contact email	Daniel.Pouly@unil.ch
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000659
     1. Label: PRIDE project
     2. Name: The circadian clock output deubiquitylating enzyme USP2-45 regulates intestinal Ca2+ absorption