Dataset Information

TMT-based phosphoproteomic analysis of WT and PKD2/3-deficient iNKT cells

ABSTRACT: Parental and PKD2/3 double-deficient DN32.D3 cells were treated with 10 ug/ml anti-CD3 and cross-linked with 100 ug/ml goat anti-Hamster IgG for 5 min. Cells were washed with ice-cold HEPES-saline and lysed with Guanidine-HCl buffer (6 M guanidine-HCl, 100 mM Tris-Cl pH8.0, 2 mM DTT). The lysates were dissolved through heating and sonication, followed by centrifugation at 20,000 × g for 15 min at 4 °C. The supernatants were reduced in 5 mM dithiothreitol at room temperature for 30 min and alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins (250 µg each) were purified using methanol–chloroform precipitation and solubilized with 25 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. The proteins were digested with 2.5 µg of Trypsin/Lys-C mix (Promega) for 16 h at 37 °C. Peptide concentrations were determined using a Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). The digested peptides (150 µg each) were labeled with 0.2 mg of TMT-10plex reagents (Thermo Fisher Scientific) for 1 h at 25 °C. After the reaction was quenched with hydroxylamine, all TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and subjected to the High-Select Fe-NTA phosphopeptide enrichment kit (Thermo Fisher Scientific). The eluates were acidified and fractionated using the Pierce High pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Nine fractions were collected using 5%, 7.5%, 10%, 12%, 14%, 16%, 18%, 20%, and 50% acetonitrile (ACN). Each fraction was evaporated using a SpeedVac concentrator and dissolved in 0.1% TFA. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC system connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 μm × 150 mm; Nikkyo Technos) with a linear gradient of 4–20% ACN for 0–150 min and 20–32% ACN for 150–190 min, followed by an increase to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with the top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375–1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, AGC target of 1e5, isolation window of 0.7 m/z, maximum injection time of 100 ms, and normalized collision energy of 32. The dynamic exclusion was set at 30 s. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer 2.2 (Thermo Fisher Scientific) with the Mascot search engine for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages, (b) precursor mass tolerance of 10 ppm, (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications, and (e) oxidation of methionine, deamidation of asparagine and glutamine, and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at a false-discovery rate (FDR) of 1% using the percolator node.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD059288 | JPOST Repository | Tue Sep 09 00:00:00 BST 2025

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	237_ishikawa_tmt10_fe_brp9_1.raw	Raw
	239_ishikawa_tmt10_fe_brp9_2.raw	Raw
	241_ishikawa_tmt10_fe_brp9_3.raw	Raw
	243_ishikawa_tmt10_fe_brp9_4.raw	Raw
	245_ishikawa_tmt10_fe_brp9_5.raw	Raw

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Publications

Invariant TCR-triggered protein kinase D activation mediates NKT cell development.

Ishikawa Eri E Kosako Hidetaka H Motooka Daisuke D Imasaka Mai M Watarai Hiroshi H Ohmuraya Masaki M Yamasaki Sho S

The Journal of experimental medicine 20250918 12

Development of invariant natural killer T (iNKT) cells in the thymus requires cell-cell interaction through invariant TCR (iTCR) and CD1d, which induces expression of the transcription factor, promyelocytic leukemia zinc finger (PLZF). However, the signaling pathway linking iTCR and PLZF remains unclear. Here, we report that a serine/threonine kinase, protein kinase D (PKD), plays a pivotal role in iNKT cell development. In T cell-specific PKD-deficient (Prkd2/3∆CD4) mice, PLZF induction and iNK ...[more]

PMID: 40965511

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