PXD075369-1

PXD075369 is an original dataset announced via ProteomeXchange.

Title	DIA analysis of FLAG-RPS19 immunoprecipitates in HCT116 cells
Description	Three independent biological replicates of HCT116 cells transiently transfected with FLAG-tagged 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). The beads were washed three times with 1 ml of HEPES-RIPA2 buffer and 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 a timsTOF HT mass spectrometer coupled to a nanoElute2 UHPLC system (Bruker). Peptides were separated on a 75 μm X 150 mm C18 reversed-phase column (Nikkyo Technos) using a segmented linear gradient: 5%–20% ACN from 0 to 40 min, 20%–35% ACN from 40 to 60 min, followed by a rapid increase to 95% ACN from 60 to 61 min, and held at 95% ACN from 61 to 75 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, an 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. DIA-MS data were analyzed using DIA-NN (version 1.9) against a human in silico spectral library. Library generation parameters included trypsin digestion with one missed cleavage allowed, 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. For DIA-NN search settings, MS1 and MS2 mass accuracies were set to auto, both neural network classifiers were configured for single-pass mode, and QuantUMS algorithm was used for quantification.
HostingRepository	jPOST
AnnounceDate	2026-03-09
AnnouncementXML	Submission_2026-03-08_18:57:34.914.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	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2026-03-08 18:57:13	ID requested
⏵ 1	2026-03-08 18:57:35	announced

Dataset with its publication pending

submitter keyword: DIA, RPS19, FLAG-IP, HCT116

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

jPOST dataset URI

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