PXD060754-1

PXD060754 is an original dataset announced via ProteomeXchange.

Title	LFQ of GFP-Trap IP-MS of BmN-4 cells expressing BmMasc-GFP
Description	BmMasc-GFP, BmMascNLS-GFP, or GFP was transiently expressed in BmN-4 cells seeded in 10 cm-diameter culture dishes. At three days post transfection, the cells were fixed with 0.1% formaldehyde, washed, and lysed on ice for 10 min in 1 mL of RIPA buffer (20 mM HEPES-KOH (pH 7.5), 1 mM EGTA, 1 mM MgCl2, 150 mM NaCl, 0.25% Na-deoxycholate, 0.05% SDS, 1% NP-40) supplemented with protease inhibitor cocktail cOmplete EDTA-free (Roche, Switzerland) and Benzonase (Merck, Germany). After centrifugation, the supernatants were incubated with GFP-Trap Magnetic Agarose (ChromoTek, Germany) for 3 h at 4 °C under gentle rotation. Proteins bound to the beads were digested by adding 200 ng of Trypsin/Lys-C mix (Promega) for 16 h at 37 °C. The digests were reduced, alkylated, acidified with trifluoroacetic acid (TFA), and desalted using a GL-Tip SDB. The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed using an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source. The peptides were separated on a 75 μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4–32% ACN gradient for 0–100 min followed by an increase to 80% ACN for 10 min and a final hold at 80% ACN for 10 min. The mass spectrometer was operated in a data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range from 375 to 1,500 m/z. Higher-energy collisional dissociation (HCD) MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against B. mori protein data supplemented with BmMasc-GFP and BmMascNLS-GFP sequences using Proteome Discoverer version 2.5 (Thermo Fisher Scientific, USA) with Sequest HT search engine. 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the percolator node and the protein FDR validator node, respectively. Label-free precursor ion quantification was performed using the precursor ions 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	2025-05-31
AnnouncementXML	Submission_2025-05-30_18:55:45.024.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: cellular organisms; NCBI TaxID: 131567;
ModificationList	L-methionine sulfoxide; Acetyl; S-carboxamidomethyl-L-cysteine
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2025-02-12 21:47:03	ID requested
⏵ 1	2025-05-30 18:55:45	announced

Dataset with its publication pending

submitter keyword: LFQ, GFP-Trap, BmMasc

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

jPOST dataset URI

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