PXD043701

PXD043701 is an original dataset announced via ProteomeXchange.

Title	LFQ of GFP-Trap IP-MS from MEF cells expressing GFP-WIPI2d
Description	Control or GFP-WIPI2d expressing MEFs were treated with or without 0.5uM Apilimod for one hour, and then cross-linked with 0.1% formaldehyde for ten mins at room temperature. After cross-linking was quenched with 100mM glycine for four mins at room temperature, cells were washed with HEPES saline containing 20mM HEPES-NaOH buffer (pH 7.5) and 137 mM NaCl, and lysed in HEPES-RIPA buffer containing 20mM HEPES-NaOH buffer (pH7.5), 150mM NaCl, 1mM EGTA, 1mM MgCl2, 0.25% (w/v) sodium deoxycholate, 0.05% SDS, 1% (v/v) NP-40, 0.2% (v/v) Benzonase Nuclease, PhosSTOP, and Protease Inhibitor Cocktail on ice. After sonication and centrifugation at 20,380 × g at 4℃ for 15 mins, the supernatants were incubated with GFP-Trap magnetic agarose beads at 4℃ for 2 h. The beads were washed four times with HEPES-RIPA buffer and then twice with 50mM ammonium bicarbonate. Proteins on the beads were digested by adding a 200 ng trypsin/Lys-C mix (Promega) at 37℃ overnight. The resultant digests were reduced, alkylated, acidified, and desalted using a GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). Liquid chromatography-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. The peptides were separated on a C18 reversed-phase column (75 μm×150 mm; Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 mins, followed by an increase to 80% ACN for ten mins and a final hold at 80% ACN for ten mins. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of three secs. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375-1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 msecs, and a normalized collision energy of 30. Dynamic exclusion was set to 20 secs. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer 2.5 with the 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 the 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 quantification was performed on the basis of the intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2024-06-25
AnnouncementXML	Submission_2024-06-25_02:57:33.295.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	L-methionine sulfoxide; Acetyl; S-carboxamidomethyl-L-cysteine
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2023-07-12 01:41:36	ID requested
⏵ 1	2024-06-25 02:57:33	announced

Dataset with its publication pending

submitter keyword: GFP-Trap, MEF, WIPI2d

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

jPOST dataset URI

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