PXD051908

PXD051908 is an original dataset announced via ProteomeXchange.

Title	Rubicon-interactome-nutrient-starvation_HeLa-Kyoto-cell
Description	HeLa Kyoto cells transiently expressing the indicated plasmids were grown in a 10-cm dish with or without starvation treatment using Earle’s Balanced Salt Solution (Sigma-Aldrich, E2888). Proteins bound to the beads were reduced with 10 mM TCEP and alkylated with 20 mM IAA. Proteins were mixed with sample/methanol/chloroform/water (1:4:1:3, v/v/v/v) and spun at 14,000 × g for 5 minutes at 30°C to separate aqueous and organic layers. After the aqueous layer was removed, the organic layer was added four times the volume of methanol and spin at 21,880 × g at 30°C for 15 minutes. Proteins removed from the supernatant were dissolved in 50 µL of 9 M urea and then 8 M urea buffer diluted with 50 mM NH4HCO3 until the concentration was <1 M urea. Proteins were digested by adding 200 ng of Trypsin Gold, Mass Spectrometry Grade (Promega) at 30ºC for 17 hours. The digests were acidified with 1% trifluoroacetic acid, and desalted using a SPE C-TIP following the procedure manual (Nikkyo Technos). The eluates were evaporated and dissolved in 15 µL of 1% trifluoroacetic acid and 2% acetonitrile. The 12 µL of samples were analyzed on an UltiMate 3000 UHPLC connected to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) through a DreamSpray ion source (AMR INCORPORATED) in data-dependent acquisition mode with the top-10 MS/MS method. MS1 spectra were measured at a resolution of 70,000, an automatic gain control target of 1e6, and a mass range from 300 to 1,500 m/z. HCD MS/MS spectra were triggered at a resolution of 17,500, an automatic gain control target of 2e5, an isolation window of 1.6 m/z, a maximum injection time of 100 milliseconds, and a normalized collision energy of 27. Dynamic exclusion was set to 20 seconds. Protein identification from raw data was performed against the SwissProt database (2017 Oct.) restricted to Homo sapiens using Sequest HT and the Mascot search engine on Proteome Discoverer 2.2 (Thermo Fisher Scientific). The search parameters were as follows: (a) trypsin as an enzyme with up to three 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; and (e) acetylation of the protein N-terminus, oxidation of methionine, deamidation of asparagine and glutamine, and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides and proteins were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed as the total peptide amount for each sample over all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2024-06-25
AnnouncementXML	Submission_2024-06-25_03:41:15.535.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	KYOSUKE YANAGAWA
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; alpha-amino acetylated residue; L-methionine sulfoxide
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2024-04-30 18:49:12	ID requested
⏵ 1	2024-06-25 03:41:15	announced

Dataset with its publication pending

submitter keyword: Rubicon-interactome

Tamotsu Yoshimori
lab head
KYOSUKE YANAGAWA
contact affiliation	Osaka University
dataset submitter

jPOST dataset URI

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