PXD028723

PXD028723 is an original dataset announced via ProteomeXchange.

Title	Label-free quantitative phosphoproteomics of ΔB-Raf:ER cells treated with U0126 or 4-HT
Description	ΔB-Raf:ER cells were treated with 10 µM U0126 or 1 µM 4-HT for 30 min. The cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication and then centrifuged. The supernatants were recovered, and proteins (200 µg each) were purified by methanol-chloroform precipitation and solubilized with 40 µL of 0.1% RapiGest SF in 50 mM TEAB. After sonication and heating, the proteins were digested with 2 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The resultant peptides were subjected to the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific), and the eluates were desalted using GL-Tip SDB (GL Sciences). The desalted eluates were evaporated in a SpeedVac concentrator and redissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through 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 0–160 min followed by an increase to 80% ACN for 10 min and finally hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 1e6, and a mass range from 350 to 1,500 m/z. MS/MS spectra were acquired at a resolution of 17,500, an AGC target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalized collision energy of 27. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer 2.4 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 0.02 Da; (d) carbamidomethylation of cysteine as a fixed modification; (e) acetylation of protein N-terminus, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at FDR of 1% using the Percolator node of Proteome Discoverer. Label-free quantification was performed based on 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	2022-03-03
AnnouncementXML	Submission_2022-09-18_04:04:45.107.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; alpha-amino acetylated residue; L-methionine sulfoxide; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2021-09-23 02:54:13	ID requested
1	2022-03-03 06:45:56	announced
⏵ 2	2022-09-18 04:04:45	announced	2022-09-18: Updated FTP location.

Yoshikawa H, Nishino K, Kosako H, Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE. J Proteomics, 258():104543(2022) [pubmed]

submitter keyword: LFQ, phosphoproteomics, ERK

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

jPOST dataset URI

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.jpostdb.org/JPST001328/