PXD042933

PXD042933 is an original dataset announced via ProteomeXchange.

Title	LFQ of Spot-Trap IP-MS from Y-79 cells expressing RD3-Spot
Description	RD3-KO Y-79 cells and RD3-SPOT-expressing cells were fixed by adding 27 µl of 37% formaldehyde (Wako) (final: 0.1%), incubated at RT for 10 min, quenched with 1 ml of 1 M Glycine-NaOH pH 7.5 (Nacalai) (final: 100 mM) at RT for 4 min, washed with 10 ml DPBS, and stocked at -80℃ as a cell pellet. For immunoprecipitation, the cell pellet was lysed by 500 µl RIPA buffer containing 5 µl Protease Inhibitor Cocktail (Nacalai) and 1 µl Benzonase Nuclease (Sigma), incubated at 4℃ for 1 hr and centrifuged at 4℃, 20000 G for 10 min to extract the supernatant. Subsequently, the supernatant was incubated with 10 µl ChromoTek Spot-Trap Magnetic Agarose (Chromotek) at 4℃ for 2 hr. The slurry was washed three times with 500 µl RIPA buffer before the reaction. After the incubation, the immunoprecipitated product was washed three times with 300 µl RIPA buffer including 3 µl of Protease Inhibitor Cocktail (Nacalai), transferred into a new 1.5 ml tube to remove non-specific binding proteins and additionally washed with 300 µl of 50 mM Ammonium Bicarbonate (ABC) buffer. The final products were resuspended with 50 µl of ABC buffer and transferred into a new 1.5ml tube. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix (Promega) at 37°C overnight. The resulting digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-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 (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 µm x 150 mm; Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 min, followed by an increase to 80% ACN for 10 min and final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an 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 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 the SwissProt database restricted to Homo sapiens using Proteome Discoverer 2.5 (Thermo Fisher Scientific) 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; (e) acetylation of 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	2024-01-27
AnnouncementXML	Submission_2024-05-09_00:07:22.036.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	L-methionine sulfoxide; Acetyl; S-carboxamidomethyl-L-cysteine
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2023-06-12 23:49:29	ID requested
1	2024-01-26 09:30:12	announced
⏵ 2	2024-05-09 00:07:23	announced	2024-05-09: Updated PubMed.

Noguchi Y, Onodera Y, Miyamoto T, Maruoka M, Kosako H, Suzuki J, vivo CRISPR screening directly targeting testicular cells. Cell Genom, 4(3):100510(2024) [pubmed]

submitter keyword: LFQ, Spot-Trap, RD3, Y-79

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/JPST002195/