PXD057876

PXD057876 is an original dataset announced via ProteomeXchange.

Title	TMTpro-18plex analysis of HEK293T cells transfected with DCAF siRNAs
Description	HEK293T cells transfected with control, CUL4, COP1, DCAF3, DCAF14, or RBBP7 siRNA were lysed in 150 µL guanidine buffer. After heating and sonication, the lysates were centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were recovered, and proteins (150 µg each) were purified by methanol-chloroform precipitation and resuspended in 20 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After sonication and heating at 95 °C for 10 min, the proteins were digested with 1.5 µg trypsin/Lys-C mix (V5072, Promega) at 37°C overnight. The digested peptides (60 µg each) were labeled with 0.5 mg TMTpro-18plex reagent (A52047, Thermo Fisher Scientific) for 1 h at 25°C. After quenching the labeling reactions with hydroxylamine, all TMT-labeled samples were pooled, acidified with TFA, and fractionated using offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific). The peptides were loaded onto a 4.6 × 250 mm Xbridge BEH130 C18 column with 3.5-µm particles (Waters) and separated using a 30-min multistep gradient of solvents A (10 mM ammonium formate, pH 9.0 in 2% ACN) and B (10 mM ammonium formate, pH 9.0 in 80% ACN), at a flow rate of 1 mL/min. Peptides were separated into 48 fractions and consolidated into 16 fractions. Each fraction was evaporated using a SpeedVac concentrator and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resulting peptides (500 ng each) was performed on an EASY-nLC 1200 UHPLC system connected to a Q Exactive Plus mass spectrometer using a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 150-mm C18 reversed-phase column with an inner diameter of 75 µm at a linear gradient of 4–20% ACN for 0–160 min and 20–32% ACN for 160–220 min, followed by an increase to 80% ACN for 10 min, and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with the top 15 MS/MS methods. The MS1 spectra were measured at a resolution of 70,000, an AGC target of 3e6, and a mass range of 375–1,400 m/z. The HCD MS/MS spectra were triggered at a resolution of 35,000, AGC target of 1e5, isolation window of 0.7 m/z, maximum injection time of 100 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.5 with the Sequest HT 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) TMTpro of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications, and (e) oxidation of methionine as a variable modification. The peptides were filtered through a percolator node at an FDR of 1%. TMT quantification was performed using a reporter ion quantifier node. Normalization was performed so that the total sum of the abundance values for each TMT channel for all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2025-11-14
AnnouncementXML	Submission_2025-11-13_07:00:13.033.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; unknown modification; unknown modification
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2024-11-13 21:17:17	ID requested
⏵ 1	2025-11-13 07:00:13	announced

Dataset with its publication pending

submitter keyword: TMT, DCAF, siRNA

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

jPOST dataset URI

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