PXD060794

PXD060794 is an original dataset announced via ProteomeXchange.

Title	DIA of CD4 SP thymocytes with or without TCR stimulation
Description	CD4 SP thymocytes were sorted and unstimulated or stimulated with α-CD3 mAb and α-CD28 mAb for 16 hours. Cells were washed with PBS and lysed in 150 μl of 6 M guanidine-HCl, 100 mM HEPES-NaOH, pH7.5, 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants were recovered, and proteins were purified by methanol–chloroform precipitation and solubilized in 20 µl of 8 M urea, 50 mM Tris-HCl, pH8.0. After sonication, the protein solutions were diluted 8-fold with 50 mM Tris-HCl, pH8.0 and digested with 1 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The resulting peptide solutions were desalted using GL-Tip SDB (GL Sciences), evaporated in a SpeedVac concentrator, and re-dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on a nanoElute 2 coupled with a timsTOF HT mass spectrometer (Bruker). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 5%–35% ACN gradient for 0–60 min followed by an increase to 95% ACN for 1 min and a final hold at 80% ACN for 4 min. Data acquisition was conducted in dia-PASEF mode with an MS1 m/z range of 100–1700 and an ion mobility (1/K0) range of 0.6–1.6. The ramp time was set to 100 ms with a 100% duty cycle. The MS2 polygon was manually defined based on the region where peptides were predominantly detected, specifically using the following four vertices: Point 1 (m/z = 300, 1/K0 = 0.64), Point 2 (m/z = 300, 1/K0 = 0.8), Point 3 (m/z = 770, 1/K0 = 0.97), and Point 4 (m/z = 770, 1/K0 = 1.15). Within the defined polygon, a 8 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 62 windows per cycle and a cycle time of 3.36 s. Protein identification was carried out in DIA-NN (version 1.9) against a mouse in silico spectral library. Parameters for constructing the library included: trypsin as the digestion enzyme, one allowed missed cleavage, peptide lengths ranging from 7 to 45 amino acids, precursor charges of 2 to 4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time and ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. In the DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).
HostingRepository	jPOST
AnnounceDate	2025-04-18
AnnouncementXML	Submission_2025-04-17_19:37:37.695.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	L-methionine sulfoxide; Acetyl; S-carboxamidomethyl-L-cysteine
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2025-02-13 18:14:00	ID requested
⏵ 1	2025-04-17 19:37:38	announced

Shinebaatar E, Morimoto J, Koga R, Nguyen TN, Sasaki Y, Yonemura S, Kosako H, Yasutomo K, Proteasome dysfunction in T cells causes immunodeficiency via cell cycle disruption and apoptosis. Int Immunol, ():(2025) [pubmed]

submitter keyword: DIA, thymocytes, proteasome, TCR

Hidetaka Kosako
lab head
Hidetaka Kosako
contact affiliation	Tokushima University
dataset submitter

jPOST dataset URI

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