PXD037179

PXD037179 is an original dataset announced via ProteomeXchange.

Title	TMTpro-18plex analysis of NTERA-2 cells treated with PROTACs
Description	NTERA-2 cells were treated with DMSO, 0.3 µM Po-P, 0.3 µM Le-P, 0.3 µM F-P, 0.3 µM Cl-P, or 0.3 µM F3C-P for 16 h in three biological replicates. The cells were lysed in 100 µL guanidine buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH, pH 7.5, 10 mM TCEP, 40 mM chloroacetamide). After heating and sonication, proteins (100 µg each) were purified by methanol–chloroform precipitation and resuspended in 20 µL 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. After sonication and heating at 95 °C for 10 min, the proteins were digested with 2 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The digested peptides (40 µg each) were labeled with 0.5 mg TMTpro-18plex reagents (Thermo Fisher Scientific) for 1 h at 25 °C. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with TFA, and fractionated by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC (Thermo Fisher Scientific) as described previously. Briefly, the peptides were loaded onto a 4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 mm particles (Waters) and separated using a 30 min multistep gradient of solvents A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN), at a flow rate of 1 mL/min. Peptides were separated into 48 fractions, which were consolidated into 16 fractions. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides (500 ng each) 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 the analytical column (75 μm × 15 cm, 3 μm; Nikkyo Technos) with a linear gradient of 4–20% for 0–115 min and 20–32% for 115–160 min, followed by an increase to 80% ACN for 10 min and finally held 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 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 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.4 with the Sequest HT search engine for identification and TMT quantification. 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) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; (e) oxidation of methionine as a variable modification. Peptides were filtered at an FDR of 1% using the Percolator node. TMT quantification was performed using the Reporter Ions Quantifier node. Normalization was performed such that the total sum of the abundance values for each TMT channel over all peptides was the same.
HostingRepository	jPOST
AnnounceDate	2023-07-22
AnnouncementXML	Submission_2023-08-21_23:50:30.387.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	2022-10-04 22:49:16	ID requested
1	2023-07-22 00:52:55	announced
⏵ 2	2023-08-21 23:50:31	announced	2023-08-22: Updated PubMed.

Yamanaka S, Furihata H, Yanagihara Y, Taya A, Nagasaka T, Usui M, Nagaoka K, Shoya Y, Nishino K, Yoshida S, Kosako H, Tanokura M, Miyakawa T, Imai Y, Shibata N, Sawasaki T, Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation. Nat Commun, 14(1):4683(2023) [pubmed]

submitter keyword: TMTpro-18plex, PROTACs

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/JPST001875/