PXD029974

PXD029974 is an original dataset announced via ProteomeXchange.

Title	Analysis of the interactomes of ZAP70, CD247 and SLP76 upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR
Description	T cells have the remarkable ability to sense and discriminate between a wide range of antigenic peptides bound to major histocompatibility complex molecules (pMHC). Here, we use interactomics to monitor in a time dependent manner the early molecular events taking place upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR. We used the mouse OT-I model in which T cells specifically recognize the ovalbumin OVA 257-264 peptide (SIINFEKL, also named N4) bound to the MHC-I molecule H-2Kb (pMHC). The OT-I TCR also recognizes altered peptide ligands amongst which SIITFEKL (T4) and SIIGFEKL (G4) with sub-optimal binding capacities (ligand affinity N4>T4>G4). For affinity purification (AP) of signalling complexes, we crossed OT-I TCR transgenic mice onto mice expressing an endogenous tagged (One-Strep-Tag: OST) version of the CD3z (also called CD247), ZAP70 or SLP76 molecules. CD8+ T cells were purified from pooled lymph nodes and spleens, shortly expanded, and were left unstimulated or stimulated with N4, T4 and G4 soluble pMHC tetramers for 30, 120 or 300 s. Complexes formed around either CD247, ZAP70 or SLP76 were then purified and analysed by mass spectrometry. Each AP-MS purification is associated with a corresponding control (purification from OT-I CD8+ T cells expressing only the endogenous version of each bait, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=3 for all time-points, and each sample was analyzed twice by single-run nano LC-MS. In addition, the whole proteome of OT-I cell was analyzed by label-free shotgun proteomics in order to calculate cellular protein abundances for each interactor.
HostingRepository	PRIDE
AnnounceDate	2022-07-05
AnnouncementXML	Submission_2022-07-05_15:52:16.551.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Anne Gonzalez de Peredo
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	phosphorylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive HF; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2021-11-26 07:52:44	ID requested
⏵ 1	2022-07-05 15:52:16	announced

Dataset with its publication pending

submitter keyword: TCR, interactome, primary CD8+ T cell, OT-I mouse, ZAP70, CD247,CD3z, SLP76, OST tag, AP-MS

Odile Schiltz
contact affiliation	Institute of Pharmacology and Structural Biology, CNRS, Toulouse, France
contact email	Odile.Schiltz@ipbs.fr
lab head
Anne Gonzalez de Peredo
contact affiliation	CNRS/IPBS
contact email	gonzalez@ipbs.fr
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD029974
     1. Label: PRIDE project
     2. Name: Analysis of the interactomes of ZAP70, CD247 and SLP76 upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR