9 cases of human cell 4D-DIA proteome_analysis data
Creators: name: Wu, Jianguang
Publisher: name: figshare
Year: 2024
Subjects: classificationCode: 310205
subject: Proteomics and metabolomics

description: Animals: SPF grade SD rats, male, around 3 months old, weight 200~250 g.Grouping (n=10): negative serum control groupResolving Blood Stasis and Penetrating Turbidity and Warming Bile Soup Containing Medicine Serum GroupPreparation method:(1) According to the conversion of rat and human equivalent dose, the standard weight of human is 60Kg, the ratio of rat and human equivalent dose is 6.17:1, then 124g (clinical dose)/60Kg×6.17=12.75g/Kg is the equivalent dose for this experiment, and the drug-containing serum is prepared by gavage with 10 times of the equivalent dose: low dose (63.75g/Kg), medium dose (127.5g/Kg), high dose (255g/Kg), and high dose (255g/Kg). (63.75g/Kg), medium dose (127.5g/Kg), and high dose (255g/Kg).(2) Acclimatization feeding for 3 days, healthy rats were randomly grouped using the random number table method; a generic dosing regimen was used, with the drug being administered twice (at 9 and 16 o'clock) or thrice a day for 3d;(3) Blood was collected 1h after the last administration: anesthetized with 0.3% sodium pentobarbital at 1ml/kg body weight, fixed in supine position, abdominal skin disinfected with 75% alcohol, abdomen opened to fully expose the abdominal aorta, and blood was collected from the abdominal aorta by a 5 ml empty needle, and the collected blood samples were allowed to stand at 4℃ for 0.5~1 hour, and then centrifuged for 10 minutes at 3000r/min to separate the serum.(4) The serum was inactivated in a constant temperature water bath at 56℃ for 30min to destroy the antibody, sterilized by 0.22μm microfilm filtration, and stored at -80℃ for spare use.<br>Cells: Human Umbilical Vein Endothelial Cells (HUVECs, Wuhan Punosai, CP-H082)Grouping: blank control group 150 μM ox-LDL + negative serum150 μM ox-LDL+drug-containing serumSample pretreatment and proteomics detection:(1) Cells were seeded in 6-well plates (about 3×105 per well), and after the cells were spread all over the plates after 72 h of culture, 1 μM adriamycin or the optimal concentration of engeletin and 200 μM huperzine II were added to incubate together for 24 h;(2) 80 μL of 10% C12Im-Cl ionic liquid with 0.1% protease inhibitor was added to the cell samples as lysis solution. A non-contact ultrasonic crusher was used for 100 s (20 s of sonication, 30 s of cooling, repeated 5 times) to extract the proteins.(3) Add DTT solution to the protein extract to a final concentration of 100 mM, denature at 95 ℃ for 5 min, transfer to a pre-wetted membrane with a molecular cut-off of 10,000, centrifuge at 16,000 g for 30 min and remove the extract; add 50 mM phosphate buffer solution to wash, centrifuge at 16,000 g for 30 min; add 50 mM IAA to the membrane and react for 30 min under light. Then add 50 mM IAA to the membrane, avoid light for 30 min, and centrifuge at 16000 g for 30 min.The membrane was washed by centrifugation with 50 mM phosphate buffer solution for 4 times, and trypsin was added into the system according to the ratio of 1:30 (enzyme/protein, w/w), and digested at 37 ℃ overnight.The peptide solution was collected by centrifugation at 16,000 g, and stored at -20 ℃ for mass spectrometry analysis.