PXD051117

PXD051117 is an original dataset announced via ProteomeXchange.

Title	Mass spectrometry-based protein interactome and phosphoproteomics data upon NRDE2 overexpression
Description	The HEK293T cells transiently expressing Flag-tagged empty vector or Flag-NRDE2-WT were lysed in NETN buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40) and immunoprecipitated with anti-Flag M2 agarose (M8823; Sigma-Aldrich, MO, USA). After washing four times with the high stringent lysis buffer, the immunoprecipitants were resolved on gradient SDS-PAGE, silver-stained, and subjected to MS sequencing and data analyses. In-solution and in-gel digestion were performed according to a previously published method (Jin et al., 2003). Briefly, gel bands were minced and destained with 50% acetonitrile in 50 mM ammonium bicarbonate. Proteins were reduced with 10 mM dithiothreitol (DTT) at 56°C, followed by alkylation with 55 mM iodoacetamide at room temperature in the dark. Trypsin digestion was performed overnight at 37°C with gentle shaking. Peptides were extracted using 1% trifluoroacetic acid in 50% acetonitrile. Samples were vacuum-dried and reconstituted in 0.1% formic acid for subsequent MS analysis. MS analysis was performed using an Easy-nLC 1200 UHPLC coupled to a QExactive HF-X mass spectrometer (Thermo Fisher Scientific, MA, USA). The tandem mass spectra were searched against the human UniProt database (Version 20140922; including 20,193 sequences) using MaxQuant (Version 1.5.3.30) (Tyanova et al., 2016). Trypsin was selected as the proteolytic enzyme, and two missed cleavages sites were allowed. Cysteine carbamidomethylation was set as the fixed modification. The oxidation of methionine (M) residues and acetylation of the protein N-terminal were set as the variable modifications. The first search mass tolerance was 20 parts-per-million (ppm), and the main search peptide tolerance was 4.5 ppm. The FDRs of the peptide-spectrum matches (PSMs) and proteins were set to less than 1%.
HostingRepository	iProX
AnnounceDate	2024-03-31
AnnouncementXML	Submission_2024-03-31_20:25:55.084.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yahui Wang
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive HF-X

Revision	Datetime	Status	ChangeLog Entry
0	2024-03-31 20:25:27	ID requested
⏵ 1	2024-03-31 20:25:55	announced

Dataset with its publication pending

submitter keyword: Interactome, phosphoproteomics, NRDE2

Gangqiao Zhou
contact affiliation	State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Proteome Research Center
contact email	zhougq114@126.com
lab head
Yahui Wang
contact affiliation	State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Proteome Research Center
contact email	wangyahui@ncpsb.org.cn
dataset submitter

iProX dataset URI