Dataset Information

Proximity labeling of proteins involved in the mechanism of excisable DD

ABSTRACT: 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.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD048060 | JPOST Repository | Sat Jan 27 00:00:00 GMT 2024

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	230707_Miyamae_DD_UbC_Tama_FusionIT_NN_PeptideGroups.txt	Txt
	230707_Miyamae_DD_UbC_Tama_FusionIT_NN_Proteins.txt	Txt
	282_Miyamae_1_DD_minus_Tama_FusionIT.raw	Raw
	284_Miyamae_2_DD_plus_Tama_FusionIT.raw	Raw
	286_Miyamae_3_DD_washout_Tama_FusionIT.raw	Raw

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Publications

Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron.

Utsugi Yuki Y Nishimura Ken K Yamanaka Satoshi S Nishino Kohei K Kosako Hidetaka H Sawasaki Tatsuya T Shigemori Hideyuki H Wandless Thomas J TJ Miyamae Yusaku Y

ACS chemical biology 20240125 2

The chemogenetic control of cellular protein stability using degron tags is a powerful experimental strategy in biomedical research. However, this technique requires permanent fusion of the degron to a target protein, which may interfere with the proper function of the protein. Here, we report a peptide fragment from the carboxyl terminus of ubiquitin as a cleavable linker that exhibits the slow but efficient cleavage of a degron tag via cellular deubiquitinating enzymes (DUBs). We designed a fu ...[more]

PMID: 38270585

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