PXD075668-1

PXD075668 is an original dataset announced via ProteomeXchange.

Title	TMT-based phosphoproteomic analysis of cardiac proto-myofibroblasts after Bcat1 knockdown
Description	Proto-myofibroblasts were treated with siRNA, washed with ice-cold HEPES-saline (20 mM HEPES-NaOH [pH 7.5], 137 mM NaCl), detached by Accutase (Nacalai Tesque), and lysed in guanidine-TCEP buffer (8 M guanidine-HCl, 100 mM HEPES-NaOH [pH 7.5], 10 mM TCEP, 40 mM CCA). After heating and sonication, 200 μg of protein was purified by methanol-chloroform precipitation and resuspended in 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. Proteins were further sonicated, heated, and digested with trypsin/Lys-C mix (Promega) for 16 hours at 37°C. Peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). Approximately 130 μg of peptides was labeled with TMTpro 16-plex reagent (Thermo Fisher Scientific) for 1 hour at 25°C. After quenching with hydroxylamine, labeled samples were pooled. Nine-tenths of the pooled sample were subjected to phosphopeptide enrichment using the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific). The enriched phosphopeptides were subsequently fractionated into 16 fractions by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific), evaporated in a SpeedVac concentrator, and dissolved in 3% acetonitrile (ACN) and 0.1% TFA. LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC coupled to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a 75 μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) using a linear gradient of 4%–20% ACN for 0–115 minutes and 20%–32% ACN for 115–160 minutes, followed by an increase to 80% ACN for 10 minutes and a final 10-minute hold at 80% ACN. The mass spectrometer was operated in data-dependent acquisition mode with a top-10 MS/MS method. MS1 spectra were acquired at 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 with an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 seconds. Raw data were analyzed against the Mus musculus SwissProt database using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine for protein identification and TMT quantification. Search parameters included trypsin with up to 2 missed cleavages, a precursor mass tolerance of 10 ppm, and a fragment mass tolerance of 0.02 Da. TMTpro labeling of lysine residues and peptide N-termini and carbamidomethylation of cysteine were set as fixed modifications, and methionine oxidation and serine/threonine/tyrosine phosphorylation were set as a variable modifications. Peptides were filtered at a false discovery rate (FDR) of 1% using the percolator node. Normalization was performed so that the total abundance across all peptides was equal for each TMT channel.
HostingRepository	jPOST
AnnounceDate	2026-07-08
AnnouncementXML	Submission_2026-07-07_15:36:13.467.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; unknown modification; unknown modification; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2026-03-16 02:09:41	ID requested
⏵ 1	2026-07-07 15:36:13	announced

Dataset with its publication pending

submitter keyword: TMT, proto-myofibroblasts, phosphorylation, Bcat1 knockdown

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

jPOST dataset URI

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