PXD068987-1

PXD068987 is an original dataset announced via ProteomeXchange.

Title	Identification of host interactors of Brucella BT4E effector proteins by Flag-tag affinity purification coupled with LC-MS/MS in HEK293T cells
Description	This study aimed to systematically identify host cellular proteins that interact with newly discovered Brucella abortus type IV secretion system (T4SS) effector proteins, designated BT4E3, BT4E4, BT4E19, BT4E32, BT4E33, BT4E37, and BT4E43. To achieve this, we employed an affinity purification–mass spectrometry (AP-MS) strategy in a human cell context. The coding sequences of individual BT4E effectors were cloned into the p3xFLAG-CMV-7.1 vector as C-terminal fusions with 3×Flag tag. These constructs, along with a p3xFLAG-CMV-7.1-control vector (mCherry-Strep only), were transiently transfected into HEK293T cells using Lipofectamine 3000. Twenty-four hours post-transfection, cells were lysed under mild non-denaturing conditions (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40) supplemented with protease and phosphatase inhibitors. Flag-tagged effector complexes were affinity-purified using Flag Sepharose beads, followed by stringent washing and elution with 2.5 mM D-desthiobiotin. Each effector purification was performed in three independent biological replicates to ensure reproducibility. Eluted protein complexes were verified by Western blotting, then processed for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS was carried out on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific) coupled online to an EASY-nLC 1000/1200 nanoflow system. The resulting proteomics data enable the identification of high-confidence host binding partners for each BT4E effector, providing insights into the molecular mechanisms by which Brucella manipulates host cellular processes—including vesicular trafficking, innate immune signaling, autophagy, and cell death pathways—to establish intracellular infection. This dataset serves as a valuable resource for understanding Brucella pathogenesis and host–pathogen interactions mediated by T4SS effectors.
HostingRepository	PRIDE
AnnounceDate	2026-02-13
AnnouncementXML	Submission_2026-02-12_23:28:02.748.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	dongjie sun
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-09-30 02:10:45	ID requested
⏵ 1	2026-02-12 23:28:03	announced

Dataset with its publication pending

ProteomeXchange project tag: Biology/Disease-Driven Human Proteome Project (B/D-HPP), Human Proteome Project, Bioinformatics Infrastructure for Life Sciences (BILS) network (Sweden)

submitter keyword: Brucella BT4E effector proteins,HEK293T,LC-MS/MS,Flag-tag affinity purification

Jiabo Ding
contact affiliation	Institute of Animal Science (IAS), Chinese Academy of Agricultural Sciences (CAAS)
contact email	dingjiabo@caas.cn
lab head
dongjie sun
contact affiliation	Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
contact email	82101211278@caas.cn
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD068987
     1. Label: PRIDE project
     2. Name: Identification of host interactors of Brucella BT4E effector proteins by Flag-tag affinity purification coupled with LC-MS/MS in HEK293T cells