PXD075368

PXD075368 is an original dataset announced via ProteomeXchange.

Title	DDA analysis of FLAG-RPS7, RPS17, and RPS19 immunoprecipitates in HCT116 cells
Description	HCT116 cells transiently transfected with FLAG-tagged RPS7, RPS17, or RPS19 expression vectors or empty vectors were fixed with 0.1% formaldehyde methanol-free (Pierce) and then lysed on ice in 1 ml of HEPES-RIPA2 buffer (20 mM HEPES-NaOH pH7.5, 1 mM EGTA, 1 mM MgCl2, 150 mM NaCl, 0.25% Na-deoxycholate, 0.05% SDS, 1% NP-40) containing multiple protease inhibitors and Benzonase (70664, Novagen). The lysates were then immunoprecipitated with anti-FLAG M2 Magnetic beads (M8823, Sigma) or anti-FLAG M2 Affinity gel (A2220, Sigma). The beads were washed three times with 1 ml of RIPA gel buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Na-deoxycholate) containing multiple protease inhibitors. Finally, the beads were washed twice with 50 mM ammonium bicarbonate. Proteins on beads were digested by addition of 200 ng trypsin/Lys-C mix (Promega) for 16 h at 37°C. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75-μm (i.d.) × 150 mm C18 reversed-phase column (Nikkyo Technos) using a linear 4–32% acetonitrile (ACN) gradient for 0–100 min, followed by an increase to 80% ACN for 10 min. The mass spectrometer was operated in a data-dependent acquisition mode with a maximum duty cycle of 3 sec. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4 × 105, and a mass range from 375 to 1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1 × 104, an isolation window of 1.6 m/z, a maximum injection time of 35 msec, and a normalized collision energy of 30. Dynamic exclusion was set to 15 sec. Raw data were analyzed directly against the SwissProt database, which was restricted to H. sapiens using Proteome Discoverer version 2.4 (Thermo Fisher Scientific), and the Sequest HT search engine. The search parameters were as follows: (a) trypsin as the enzyme, with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the percolator node. Label-free precursor ion quantification was performed using the precursor ion quantifier node, and normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2026-03-09
AnnouncementXML	Submission_2026-03-08_18:32:02.945.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	L-methionine sulfoxide; Acetyl; S-carboxamidomethyl-L-cysteine
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2026-03-08 18:24:09	ID requested
⏵ 1	2026-03-08 18:32:03	announced

Dataset with its publication pending

submitter keyword: DDA, RPS7, RPS17, RPS19, FLAG-IP, HCT116

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

jPOST dataset URI

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