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Add an appropriate amount of lysis buffer (PBS solution containing a final concentration of 1% PMSF), sonicate in an ice bath, centrifuge at 12000g for 10 min at 4°C, and extract the supernatant; add an equal volume of 100% ACN solution, mix well, and place on ice for 1h; centrifuge at 10000g for 10 min after precipitation is complete, and extract all supernatant except the precipitate; freeze and drain until the volume After half of the volume was reduced, the sample was centrifuged at 10,000g with pre-wetted 10 kD ultrafiltration tubes (Merck Millipore, UFC501096, Germany) at 4°C for 20 min, and then the above operation was repeated with 200 µL of ultrapure water, and the penetration solution was collected. Bradford method to determine the protein concentration. Take 20 μL of diluted BSA solution into the enzyme standard plate, and set up 3 replicate wells for each concentration. Add 20 μL of the diluted protein sample to the ELISA plate, and set up 3 replicate wells for each sample. Add 180 μL of G250 color development solution to each well, and develop the color at room temperature and avoid light for 5 min. Determine the absorbance value of A580 by ELISA. The concentration of protein samples was calculated according to the standard curve and the dilution of protein samples.Take 100 μg of protein and dissolve it to about 1 μg/μL using U2 lysis buffer. Add 100 mM TEAB and dilute the protein 6 times, then add 0.5M CaCl2 and shake and centrifuge. Add trypsin for enzymatic digestion (trypsin: protein = 1:100) and incubate at 37°C for more than 8h. Weigh 10mg of C18 column material, corresponding to each 100μg peptide sample. 1mL of methanol activated column material, shake and centrifuge, discard supernatant. Add 1mL of 0.1% FA acidified, centrifuge and discard supernatant. Peptide samples were acidified with an equal volume of 0.1% FA, shaken, vortexed and added to a centrifuge tube, mixed silently for 30 min, centrifuged and supernatant discarded. The samples were then washed twice with 0.1% FA + 3% ACN for desalting and eluted with 1mL of 0.1% FA + 80% ACN. The eluted peptides were dried with a vacuum concentrator.Dissolve peptide samples with 20 μL dissolution buffer (0.5M TEAB), add 70 μL isopropanol, and shake and centrifuge. Label according to the iTRAQ-8 labeling kit (SCIEX) instructions, with labels 113, 114 and 115 for the MCD group and 116, 117 and 118 for the ACT001-H group. After the samples were labeled and mixed, the mixed peptides were then separated by applying the Ultimate 3000 HPLC system (Thermo DINOEX, USA) for graded separation of the peptide samples. The chromatographic column used was a Welch C18 column (250 × 4.6 mm 5mm). The peptides were separated using a gradual increase in ACN concentration under alkaline conditions, combined into 12 fractions, and the combined fractions were desalted on a Strata-X column and dried under vacuum.The peptide samples were uploaded to the machine buffer diluted to 1 μg/μL, and the loading volume was set to 8 μL for 90 min in scanning mode. Peptides with mass-to-charge ratios in the range of 350-1200 were scanned in the samples. The mass spectrometry data acquisition was performed using a Triple TOF 5600 + liquid mass spectrometry system (AB SCIEX, USA). Peptide samples were dissolved in 2% acetonitrile/0.1% formic acid and analyzed using a Triple TOF 5600 plus mass spectrometer coupled to an Eksigent nanoLC system (AB SCIEX, USA). The peptide solution was added to a C18 capture column (3 µm, 350 µm × 0.5 mm, AB Sciex, USA) and gradient eluted on a C18 analytical column (3 µm, 75 µm × 150 mm, Welch Materials, Inc) with a 90 min time gradient at a flow rate of 300 nL/min. The two mobile phases were buffer A (2% acetonitrile/0.1% formic acid/ 98% H2O) and buffer B (98% acetonitrile/0.1% formic acid/ 2% H2O). For IDA (information-dependent acquisition), primary mass spectra were scanned with an ion accumulation time of 250 ms, and secondary mass spectra of 30 precursor ions were acquired with an ion accumulation time of 50 ms. MS1 spectra were acquired in the range of 350-1200 m/z, and MS2 spectra were acquired in the range of 100-1500 m/z. The dynamic exclusion time of precursor ions was set to 15 s."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["The mass spectrometry downstream data were searched for databases using ProteinPilot and analyzed for iTraq quantification. The database used in this case was the UniProt database (http://www.uniprot.org/). DEPs were identified using a fold change (FC) threshold, with p values <0.05 considered significant after adjustment, FC ≥1.5 for expression upregulation and FC ≤0.67 for expression downregulation. GO annotations (http://www.geneontology.org/) were performed on selected DEPs to classify them functionally according to three major terms: biological process, cellular component and molecular function, and Fisher's exact test was applied to identify GO entries that were significantly enriched for DEPs. KEGG annotation (http://www.genome.jp/kegg/) was performed to identify the most significant biochemical metabolic pathways and signal transduction pathways in which DEPs are involved."],"omics_type":["Proteomics"],"labhead":["Ping Li"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Department of Hepatology, Tianjin Second People’s Hospital, Tianjin, China. Tianjin Research Institute of Liver Diseases, Tianjin, China."],"species":["Mus Musculus (mouse)"],"submitter_mail":["zh93002929@163.com"],"publication":["37443174 Zhou H, Niu B, Wu X, Chu W, Zhou Y, Chen Z, Mi Y, Liu Y, Li P. iTRAQ-based quantitative proteomics analysis of the effect of ACT001 on non-alcoholic steatohepatitis in mice. Sci Rep. 2023 13(1):11336 10.1038/s41598-023-38448-4"],"submitter_affiliation":["Tianjin Medical University"],"submitter_country":["China"],"pubmed_abstract":["ACT001 is a novel sesquiterpene lactone derivative that has been shown to have significant antitumor and anti-inflammatory effects. However, the effect of ACT001 on nonalcoholic steatohepatitis (NASH) is unknown. Methionine and choline deficient (MCD) diet induced NASH model in C57BL/6J mice. Steatosis, inflammation and fibrosis-related indices of serum and liver tissues were detected by fully automated biochemical analyzer, enzyme-linked immunosorbent assay (ELISA) kit, flow cytometry, hematoxylin and eosin (H&E), Masson and immunohistochemical staining. The results showed that ACT001 reduced serum lipid and inflammatory factor levels, attenuated hepatic steatosis, inflammation and fibrosis, and inhibited hepatic oxidative stress and activation of NOD-like receptor protein 3 (NLRP3) inflammatory vesicles in NASH mice. In addition, 381 differentially expressed proteins (DEPs), including 162 up-regulated and 219 down-regulated proteins, were identified in the MCD group and ACT001 high-dose group using isotope labeling relative and absolute quantification (iTRAQ) technique analysis. Among these DEPs, five proteins associated with NAFLD were selected for real-time fluorescence quantitative PCR (RT-qPCR) validation, and the results were consistent with proteomics. In conclusion, ACT001 has a therapeutic effect on NASH, and the results of proteomic analysis will provide new ideas for the mechanism study of ACT001 for NASH treatment."],"pubmed_title":["iTRAQ-based quantitative proteomics analysis of the effect of ACT001 on non-alcoholic steatohepatitis in mice."],"pubmed_authors":["Zhou Hui H, Niu Bin B, Wu Xue X, Chu Weike W, Zhou Yibing Y, Chen Ze Z, Mi Yuqiang Y, Liu Yonggang Y, Li Ping P"],"additional_accession":[]},"is_claimable":false,"name":"iTRAQ-Based Quantitative Proteomics Analysis of the Effect of ACT001 on Non-alcoholic Steatohepatitis in Mice","description":"Non-alcoholic steatohepatitis (NASH) is considered a progressive stage of non-alcoholic fatty liver disease (NAFLD) that can progress to cirrhosis and hepatocellular carcinoma. ACT001 is a novel sesquiterpene lactone derivative that has been shown to have significant antitumor and anti-inflammatory effects. However, the effect of ACT001 on NASH is unclear.Methionine- and choline-deficient (MCD) diet was fed to C57BL/6J mice for 6 weeks to induce the NASH model.iTRAQ quantitative proteomic analysis was performed to clarify the expression of differential proteins (DEPs) after ACT001 action.381 DEPs, including 162 up-regulated and 219 down-regulated proteins, were identified between the model and ACT001 high-dose groups.the function of DEPs in the proteome and the signaling pathways involved will provide new ideas for the research of new drugs for NASH treatment.","dates":{"publication":"2026-08-17","submission":"2022-08-08"},"accession":"PXD035890","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:57577","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:77173","NEWT:112509","NEWT:475174","NEWT:111205","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:294671","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:1907535","NEWT:135622","NEWT:4615","NEWT:67352","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:111225","NEWT:7719","NEWT:29073","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:29064","NEWT:108061","NEWT:60711","NEWT:2020713","NEWT:224308","NEWT:273791","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:33554","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:37919","NEWT:2579791","NEWT:171719","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:1538","NEWT:2065263","NEWT:199225","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:980415","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:66440","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:34878","NEWT:10886","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:1122981","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:1385720","NEWT:119562","NEWT:1883446","NEWT:31345","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:1945864","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:133894","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT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