PXD065091

PXD065091 is an original dataset announced via ProteomeXchange.

Title	DIA of GFP-Trap IP from worms expressing ROOM1/2-GFP
Description	Worms containing the transgene room-1::FLAG::GFP or room-2::FLAG::GFP were used for the Co-IP MS experiments. Approximately 25,000 gravid adult hermaphrodites were collected and homogenized in HBS buffer (20 mM HEPES-NaOH pH7.5, 150 mM NaCl, 1 mM MgCl2, and 1 mM EDTA with Complete Ultra Tablet Mini, EDTA-free (Roche)). The homogenates were crosslinked with 0.2% paraformaldehyde and solubilized in HEPES-RIPA buffer (20 mM HEPES-NaOH pH7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 0.05% SDS with Complete Ultra Tablet Mini, EDTA-free). IP was performed using GFP-Trap magnetic agarose (Chromotek), and proteins bound to the beads were digested with 200 ng trypsin/Lys-C mix (Promega) at 37 ºC overnight. The resulting peptides were reduced, alkylated, acidified, and desalted using the GL-Tip SDB (GL Sciences). The eluates were evaporated using a SpeedVac concentrator and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed using a timeTOF HT mass spectrometer (equipped with a CaptiveSpray2 ion source) coupled with a nanoElute2 UHPLC system (Bruker). Peptides were separated on a 75 μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos). Mobile phase A consisted of ultrapure water containing 0.1% formic acid while mobile phase B consisted of acetonitrile containing 0.1% formic acid. The gradient was initiated with 5% solvent B, increased to 35% at 60 min and 95% at 61 min, and finally held at 95% for 65 min. Data acquisition was conducted in the 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 the peptides were predominantly detected using the following four vertices: Point1 (m/z = 300, 1/K0 = 0.6), Point2 (m/z = 300, 1/K0 = 0.8), Point3 (m/z = 968, 1/K0 = 1.1), and Point4 (m/z = 968, 1/K0 = 1.3). Within the defined polygon, a 22 Da isolation window was employed with an overlap of 0.5 Da for each window, resulting in 31 windows per cycle and a cycle time of 1.58 s. DIA-MS data were analyzed using DIA-NN (version 1.9) against an in silico predicted spectral library constructed from the C. elegans WormBase protein database (release WS285). Library generation parameters included trypsin digestion, with one missed cleavage allowed, peptide lengths ranging from 7–45 amino acids, precursor charges from 2–4, and a fragment ion m/z range of 200–1800. Features such as library-free search, deep learning-based retention time, ion mobility predictions, N-terminal methionine cleavage, and cysteine carbamidomethylation were enabled. The MS1 and MS2 mass accuracies in the DIA-NN search settings were set to auto, both neural network classifiers were configured for single-pass mode, and the quantification strategy was set to QuantUMS (high precision).
HostingRepository	jPOST
AnnounceDate	2026-04-09
AnnouncementXML	Submission_2026-04-08_19:48:33.468.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Caenorhabditis elegans; NCBI TaxID: 6239;
ModificationList	S-carboxamidomethyl-L-cysteine; unknown modification
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2025-06-16 22:19:06	ID requested
⏵ 1	2026-04-08 19:48:33	announced

Dataset with its publication pending

submitter keyword: DIA, GFP-Trap, ROOM-1/2

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

jPOST dataset URI

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