PXD057768

PXD057768 is an original dataset announced via ProteomeXchange.

Title	Methylcobalamin protects against liver failure via engaging gasdermin E
Description	To probe the binding sites of MeCbl on GSDME, 0.4 μM recombinant human GSDME (CSB-EP006766HU) purchased from CUSABIO (Wuhan, China) was incubated with 10 μM MeCbl in 100 μL buffer (20 mM HEPES, 150 mM NaCl and 50 μM DPA) at room temperature for 2 h and protected from light. 0.4 μM recombinant human GSDME was incubated with ddH2O as negative control, or with HOCbl for binding sites analysis between GSDME and cobalamin. All groups were equally prepared ten incubation samples and mixed for further analysis. Trypsin and Glu C were then added in the mixture overnight, and formic acid was added to adjust the pH down to three to terminate the enzyme digestion. Activated the C18 desalting column with 100 μL 100% acetonitrile, then centrifuged at 400 g for 3 min. Added 100 μL of 0.1% formic acid and centrifuged at 400 g for 3 min for column equilibration. Replaced with new EP tube, added samples to column, and centrifuged at 400 g for 3 min. Washed the column twice with 100 μL of 0.1% formic acid and centrifuged at 400 g for 3 min, washed once again with 100 μL water at pH10. The eluents were collected with 70% acetonitrile, each sample was lyophilized and stored at -80°C until further mass spectrometry analysis. Prepared the mobile phase solution, A (0.1% (v/v) formic acid in ddH2O), B (0.1% (v/v) formic acid and 80% acetonitrile in ddH2O), the lyophilized samples were dissolved with 10 µL solution A, then centrifuged at 14000 g for 20 min at 4°C, 400 ng supernatant of the samples were injected onto a Thermo Scientific™ UltiMate™ 3000 UHPLC coupled to a timsTOF HT mass spectrometer (BRUKER). Peptides were then separated over a 100 μm ⅹ15 cm ReproSil-Pur C18-AQ 1.5-µm silica beads (Beijing Qinglian Biotech Co.,Ltd, Beijing, China), at a flow rate of 300 nL/min using a gradient: 0-4 min (5-10% B), 4-46 min (10-24% B), 46-53 min (36% B), 53-54 min (95% B), 54-60 (95% B). The timsTOF HT mass spectrometer was used for liquid quality detection with Captive Spray source, and the mass spectrum was collected in DDA mode. The scanning range of the mass spectrum was m/z 100-1700, the primary mass spectrum resolution was set to 60,000 (1222 m/z), and the cumulative time was set to 100 ms in the TIMS tunnel. The capillary voltage is set at 1.6 kV and the mobility is 0.6-1.6 cm2/(V). The total cycle time was 1.1s with 10 PASEF cycles. The mass spectrometry raw data was processed by FragPipe to search against the target protein database. The precursor ion mass tolerance was ±15 ppm; the fragment mass tolerance was ±0.02 Da. The static modification was carbamidomethylation of cysteine; the dynamic modifications were oxidation of methionine, acetylation of N-termini of peptides and C63H91CoN13O14P or C62H88CoN13O14P of cysteine; a maximum of two missed cleavages were allowed.
HostingRepository	iProX
AnnounceDate	2024-11-11
AnnouncementXML	Submission_2024-11-11_19:01:39.981.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Wanfeng Xu
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	timsTOF Pro

Revision	Datetime	Status	ChangeLog Entry
0	2024-11-11 19:01:14	ID requested
⏵ 1	2024-11-11 19:01:40	announced

Dataset with its publication pending

submitter keyword: Peptide mapping , Mass spectrometry

Haiping Hao
contact affiliation	China Pharmaceutical University
contact email	haipinghao@cpu.edu.cn
lab head
Wanfeng Xu
contact affiliation	China Pharmaceutical University
contact email	15251768808@163.com
dataset submitter

iProX dataset URI