PXD072084

PXD072084 is an original dataset announced via ProteomeXchange.

Title	Proteomic profiling of the nucleus accumbens and ventral tegmental in rats across social hierarchy
Description	Mass spectrometry sample collection Sprague–Dawley rats were sacrificed by cervical dislocation. Brain regions (NAc and VTA) were dissected and stored at –80 °C until use. Samples were retrieved from –80 °C storage and lysed with lysis buffer (4x volume of the sample; 8 M urea solution, 1% protease inhibitor cocktail) followed by sonication. The lysates were centrifuged at 12,000g for 10 minutes at 4 °C to remove cellular debris. Supernatants were transferred to new tubes and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, #23227). For each sample, 5 mg of brain tissue (NAc or VTA) was used, obtained from a pool of 2 rats. An equal amount of protein from each sample was used for enzymatic digestion. The volumes were equalized using lysis buffer. Pre-chilled acetone was added in a 1:1 ratio, then mixed using a vortexer before pre-chilled acetone was added (4x volume of the sample). The samples were then incubated at –20 °C for 2 hours for protein precipitation. After centrifugation at 4500g for 5 minutes, the supernatants were discarded, and the protein pellets were washed twice with pre-chilled acetone. The pellets were air-dried, and then resuspended in tetraethylammonium bromide buffer with a final concentration of 200 mM. The resuspended proteins were sonicated to disrupt any remaining aggregates. Trypsin was added at a ratio of 1:50 (enzyme:protein, w/w) and the samples were incubated overnight for protein digestion. Dithiothreitol (DTT) was then added to achieve a final concentration of 5 mM, and the samples were incubated at 56 °C for 30 minutes for reduction. Subsequently, iodoacetamide (IAA) was added to achieve a final concentration of 11 mM and the samples were incubated at room temperature in the dark for 15 minutes for alkylation. Mass spectrometry proteomic analysis Each sample was fractionated using high pH reverse-phase high performance liquid chromatography (HPLC) with an Agilent 300 Extend C18 column (particles, 5 μm; internal diameter 4.6 mm; length, 250 mm). Briefly, peptides were separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 10 over 80 min into 80 fractions. Then, peptides were combined into 9 fractions and dried by vacuum centrifuging. Subsequently, they were placed into an Orbitrap Exploris™ 480 mass spectrometer for analysis. The ion source voltage was set to 2.3 kV, and the FAIMS compensation voltage (CV) was set to –45 V and –65 V. Both precursor ions and their secondary fragments were detected and analyzed using the high-resolution Orbitrap. The primary mass spectrometry scan range was set to 400-1200 m/z with a resolution of 60,000. The secondary mass spectrometry scan range was set with a fixed starting point of 110 m/z and a resolution of 15,000, with TurboTMT turned off. Data acquisition was performed using a data-dependent acquisition (DDA) program based on cycle time. Within a 1.0 s cycle, precursor ions were selected for fragmentation based on their signal intensity from high to low and subjected to HCD fragmentation with 27% collision energy in the HCD collision cell, followed by sequential secondary mass spectrometry analysis. To maximize mass spectrometer efficiency, the automatic gain control (AGC) was set to 100%, the signal threshold was set to 5×104 ions/s, the maximum injection time was set to auto mode, and the dynamic exclusion time for tandem mass spectrometry scans was set to 20 s to avoid repeated scanning of precursor ions. MS data processing and bioinformation analysis The resulting MS/MS data were processed using the MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched against the human SwissProt database (20422 entries) concatenated with a reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in first search and 5 ppm in main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification, and acetylation on protein N-terminal and oxidation on Met were specified as variable modifications. FDR was adjusted to < 1%. Pathway membership information was obtained from KEGG pathways and categorical GO annotation supplied werebiological process (BP), molecular function (MF), and cellular component (CC). For further hierarchical clustering based on differentially expressed protein functional classification (such as: GO, Domain, Pathway, Complex), we first collated all the categories obtained after enrichment along with their P values, and then filtered for those categories that were at least enriched in one of the clusters with a P value of <0.05. This filtered P value matrix was transformed by the function x = −log10 (P value). These p values were then clustered by one-way hierarchical clustering (Euclidean distance, average linkage clustering). Cluster membership was visualized by a heatmap plotted using the ggplot2 (version 3.3.5) and pheatmap (version 1.0.12) R-package. All differentially expressed protein database accessions or sequences were searched against the STRING database version 11.5 for protein-protein interactions. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. STRING defines a metric called “confidence score” to define interaction confidence; we fetched all interactions that had a confidence score ≥ 0.7 (high confidence). The interaction network from STRING was visualized in R package “networkD3”
HostingRepository	iProX
AnnounceDate	2025-12-17
AnnouncementXML	Submission_2026-04-30_00:58:15.400.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Jiafeng Zhong
SpeciesList	scientific name: Rattus norvegicus; NCBI TaxID: 10116;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2025-12-17 01:32:59	ID requested
⏵ 1	2026-04-30 00:58:15	announced

Dataset with its publication pending

submitter keyword: social hierarchy, the nucleus accumbens, ventral tegmental

Yinjie Zhu
contact affiliation	Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences
contact email	yj.zhu1@siat.ac.cn
lab head
Jiafeng Zhong
contact affiliation	Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences
contact email	jf.zhong@siat.ac.cn
dataset submitter

iProX dataset URI