PXD044493

PXD044493 is an original dataset announced via ProteomeXchange.

Title	Mouse Neuroblastoma Cells Proteome by LC-MS/MS
Description	N2a cells were transfected with pCAGGS vector or pCAGGS-RABV-M (expressing rabies virus matrix protein) for 36 hours, cells were lysed for SDS-PAGE. The area where the proteins were located in the SDS-PAGE was scooped and cut into small pieces of about 1 mm3; the decolorization solution (15 mM K3Fe(CN)6/50 mM Na2S2O3) was added and shaken until the gelatin block was decolorized, and the supernatant was removed, the decolorization reaction was terminated by adding double-distilled water to submerge the gelatin block, and the washings were aspirated after shaking for 10 min; the gelatin block was submerged with a 50% ACN/100 mM NH4HCO3 ( pH 8.0) solution was used to submerge the gel block, shaking for 10 min, aspirating the washings, and repeating the process for a total of three times; 100% ACN was added to submerge the gel block, shaking for 10 min, aspirating the washings, and then the gel block was dried up in a vacuum extractor; 10 mM DTT/50 mM NH4HCO3 (pH 8.0) solution was added to the gel block, and the gel block was incubated at 56°C for 1h for the reduction reaction, followed by aspirating the The leachate was then added 55 mM iodoacetamide/50 mM NH4HCO3 (pH 8.0) solution, and the reaction was carried out at room temperature in the dark for 30 min, after which the leachate was aspirated; 100% ACN was added, shaken for 20 min, the leachate was aspirated, and the gelatin block was drained; a suitable amount of trypsin was added to the gelatin block, and 50 mM NH4HCO3 solution was added to completely cover the gelatin block Then add 60% ACN/5% formic acid as extraction solution, ultrasonic shaking for 10 minutes, centrifuge and aspirate the supernatant into a new centrifuge tube; repeat the extraction process twice, combine the extraction solution, and drain it in a centrifugal concentrator; desalinate the peptide using a C18 column, and freeze at -20 ℃ to wait for the detection of the machine. Mass spectrometry analyses were performed using a Q Exactive Plus liquid mass spectrometry system from Thermo. Samples were separated by a nanoliter flow rate liquid phase UltiMate 3000 RSLCnano system. Peptide samples were solubilized in the upwelling buffer, inhaled by an autosampler and bound to a C18 capture column (3 μm, 120 Å , 100 μm × 20 mm), followed by elution to an analytical column (2 μm, 120 Å, 75 μm × 150 mm) for separation. An analytical gradient was established using two mobile phases (mobile phase A: 3% DMSO, 0.1% formic acid, 97% H2O and mobile phase B: 3% DMSO, 0.1% formic acid, 97% ACN). The flow rate of the liquid phase was set to 300 nL/min. For mass spectrometry DDA mode analysis, each scan cycle consisted of one full MS scan (R = 70 K, AGC = 3e6, max IT = 20 ms, scan range = 350-1800 m/z) and 15 subsequent MS/MS scans (R = 17.5 K, AGC = 2e5, max IT = 20 ms). AGC = 2e5, max IT = 100 ms). the HCD collision energy was set to 28. the screening window for the quadrupole was set to 1.6 Da. the dynamic exclusion time for repeated ion acquisitions was set to 35 s. Mass spectrometry data generated by Q Exactive Plus were searched by MaxQuant (V1.6.2.10) using the database search algorithm MaxLFQ.The database used for the search was the Mouse Proteome Reference Database.
HostingRepository	iProX
AnnounceDate	2023-08-09
AnnouncementXML	Submission_2024-03-18_20:12:43.594.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yueming Yuan
SpeciesList	scientific name: Mus musculus; NCBI TaxID: 10090;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2023-08-10 20:22:01	ID requested
1	2023-08-10 20:22:09	announced
⏵ 2	2024-03-18 20:12:44	announced	2024-03-19: Update information.

Yuan Y, Fang A, Wang H, Wang C, Sui B, Zhao J, Fu ZF, Zhou M, Zhao L, Lyssavirus M protein degrades neuronal microtubules by reprogramming mitochondrial metabolism. mBio, 15(3):e0288023(2024) [pubmed]

submitter keyword: N2a cells, Proteome profiling, Rabies virus matrix protein transfection

Ling Zhao
contact affiliation	School of Animal Medicine, Huazhong Agricultural University
contact email	lingzhao@mail.hzau.edu.cn
lab head
Yueming Yuan
contact affiliation	School of Animal Medicine, Huazhong Agricultural University
contact email	18086188238@163.com
dataset submitter

iProX dataset URI