PXD048060-1

PXD048060 is an original dataset announced via ProteomeXchange.

Title	Proximity labeling of proteins involved in the mechanism of excisable DD
Description	Excisable DD construct was fused with TurboID and transduced into NIH3T3 cells. The cells were exposed to Shield-1 (1 μM) for 24 h, followed by co-treatment of Shield-1 (1 μM) and biotin (50 µM) for 24 h (biological replicates; n = 3). The cell lysates in guanidine-TCEP buffer 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 were purified by methanol–chloroform precipitation and solubilized in PTS buffer (12 mM SDC, 12 mM SLS, 100 mM Tris-HCl, pH 8.0). After sonication and heating, the protein solution was diluted 5-fold with 100 mM Tris-HCl, pH 8.0 and digested with 1:100 w/w of trypsin at 37°C overnight. After heating, the resulting peptide solutions were diluted 2-fold with TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Biotinylated peptides were captured on a 15 µL slurry of MagCapture HP Tamavidin 2-REV magnetic beads after incubation for 3 h at 4°C. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 1 mM biotin in TBS for 15 min at 37°C twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% trifluoroacetic acid and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer using a nanoelectrospray ion source. The peptides were separated on a 150-mm C18 reversed-phase column with an inner diameter of 75 µm (Nikkyo Technos) using a linear 4–32% acetonitrile gradient for 0–60 min, followed by an increase to 80% acetonitrile for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. The MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375–1,500 m/z. HCD MS/MS spectra were acquired in a linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 200 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 10 s. Raw data were analyzed directly against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer version 2.5 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification, and (e) acetylation of protein N-terminus, oxidation of methionine, and biotinylation of lysine as variable modifications. Peptides were filtered at a false discovery rate (FDR) of 1% using the Percolator node. Label-free quantification was performed based on the intensities of the precursor ions using a precursor ion quantifier node.
HostingRepository	jPOST
AnnounceDate	2024-01-27
AnnouncementXML	Submission_2024-01-26_09:29:08.763.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; Acetyl; N6-biotinyl-L-lysine; L-methionine sulfoxide
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2023-12-21 17:55:13	ID requested
⏵ 1	2024-01-26 09:29:09	announced

Utsugi Y, Nishimura K, Yamanaka S, Nishino K, Kosako H, Sawasaki T, Shigemori H, Wandless TJ, Miyamae Y, Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron. ACS Chem Biol, 19(2):497-505(2024) [pubmed]

submitter keyword: TurboID, destabilizing domain (DD)

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

jPOST dataset URI

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