Dataset Information

DIA of CD4 SP thymocytes with or without TCR stimulation

ABSTRACT: 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).

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD060794 | JPOST Repository | Fri Apr 18 00:00:00 BST 2025

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	PJ1381_Morimoto_10_KI_TCR_timsHT_DIA_Slot2-38_1_2766.d.zip	Other
	PJ1381_Morimoto_11_KI_TCR_timsHT_DIA_Slot2-42_1_2774.d.zip	Other
	PJ1381_Morimoto_12_KI_TCR_timsHT_DIA_Slot2-46_1_2782.d.zip	Other
	PJ1381_Morimoto_13_KIKO_TCR_timsHT_DIA_Slot2-35_1_2760.d.zip	Other
	PJ1381_Morimoto_14_KIKO_TCR_timsHT_DIA_Slot2-39_1_2768.d.zip	Other

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Publications

Proteasome dysfunction in T cells causes immunodeficiency via cell cycle disruption and apoptosis.

Shinebaatar Erkhembayar E Morimoto Junko J Koga Rinna R Nguyen Thanh Nam TN Sasaki Yuki Y Yonemura Shigenobu S Kosako Hidetaka H Yasutomo Koji K

International immunology 20250701 8

Proteasomes are essential molecular complexes that regulate intracellular protein homeostasis by selectively degrading ubiquitinated proteins. Genetic mutations in proteasome subunits lead to proteasome-associated autoinflammatory syndromes (PRAAS) characterized by autoinflammation, partial progressive lipodystrophy, and, in certain cases, immunodeficiency. However, the molecular mechanisms by which proteasome dysfunction results in these phenotypes remain unclear. Here, we established a mouse m ...[more]

PMID: 40223594

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