PXD028626

PXD028626 is an original dataset announced via ProteomeXchange.

Title	TMTpro 16-plex analysis of siRAD23B and siControl in replicatively and ETP-treated senescent cells
Description	Replicatively-senescent and ETP-treated senescent WI-38 cells were transfected with siRNAs (Stealth Select RNAi RAD23B siRNA #1 and siGENOME non-targeting control siRNA #2) using Lipofectamine RNAiMAX in four biological replicates. At 72 h after transfection, the cells were washed twice with cold-PBS and lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH7.5), 10 mM Tris (2-carboxyethyl) phosphine, and 40 mM chloroacetamide. The lysates were dissolved by heating and sonication and then centrifuged at 20,000 g for 15 min at 4°C. The supernatants were recovered, and proteins (40 µg each) were purified by methanol-chloroform precipitation and solubilized by 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. The proteins were digested with 0.5 µg trypsin/Lys-C mix (Promega) at 37°C overnight. Peptides (12.5 µg) from each sample were labeled with 0.25 mg of TMTpro 16-plex reagents (Thermo Fisher Scientific) and combined and fractionated using the Pierce high pH reversed-phase peptide fractionation kit. Nine fractions were collected using 5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% ACN. LC-MS/MS analysis of the resultant peptides (1 µg) from each fraction except the first was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75-μm-inner-diameter, 150-mm C18 reversed-phase column (Nikkyo Technos) with a linear gradient of 4–24% for 0–140 min and 24–36% for 140–180 min, followed by an increase to 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a top 15 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from 375 to 1,400 m/z. MS/MS spectra were triggered at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.4 m/z, a maximum injection time of 100 ms, and a normalized collision energy of 31. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer (v.2.4) with Sequest HT search engine for identification and TMT quantification. The search parameters were the following: (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) TMTpro of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) oxidation of methionine as a variable modification. Peptides were filtered at a false discovery rate <0.01 using the Percolator node. TMT quantification was performed using the Reporter Ions Quantifier node.
HostingRepository	jPOST
AnnounceDate	2023-07-22
AnnouncementXML	Submission_2023-08-21_23:51:15.697.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	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; unknown modification; unknown modification
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2021-09-18 08:40:31	ID requested
1	2023-07-22 00:48:50	announced
⏵ 2	2023-08-21 23:51:16	announced	2023-08-22: Updated PubMed.

Iriki T, Iio H, Yasuda S, Masuta S, Kato M, Kosako H, Hirayama S, Endo A, Ohtake F, Kamiya M, Urano Y, Saeki Y, Hamazaki J, Murata S, Senescent cells form nuclear foci that contain the 26S proteasome. Cell Rep, 42(8):112880(2023) [pubmed]

submitter keyword: TMTpro 16-plex, RAD23B, replicative senescence, ETP-induced senescence

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

jPOST dataset URI

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