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ezie.itRoberto StellaData-dependent acquisitionMass SpectrometryBottom-up proteomicsMagnolia officinalis (l.)Oxidative stressNeuroprotectionLc-hrms/msPhytochemicalsRatLabel-free proteomicsAge-related disor-dershttps://www.ebi.ac.uk/pride/archive/projects/PXD075711BrainRat brain slices were lysed in ice-cold buffer containing 8 M urea, 2% (w/v) SDS, 100 mM Tris-HCl, pH 8, and a protease inhibitors cocktail and stored at -80 °C until use (n=5 for each condition tested). A quality control (QC) sample was prepared by mixing equal amounts of protein ex-tracts derived from brain slices and divided in 3 independent aliquots. For the analysis, lysates of brain slices and QC samples were processed following the filter-aided sample preparation (FASP) method. 50 µg of total proteins were diluted in 200 µL of 8 M urea in 50 mM Tris-HCl, pH 8, loaded into filters with a molecular weight cut-off of 10 kDa, and centrifuged (15 min, 14,000 g, 25 °C). Reduction of disulfide bonds was performed by adding 200 µL of 25 mM DTT, 50 mM Tris-HCl (45 min at 55 °C), and after centrifugation, alkylation of cysteines occurred by incubating filters in an equal volume of 50 mM Tris-HCl containing 55 mM iodoacetamide (45 min in the dark at room temperature). After centrifugation (15 min, 14,000 g, 25 °C), filters were incubated (18 h, 37 °C) in presence of 2 µg of sequencing grade modified trypsin in solution (100 μL in 50 mM Tris-HCl, 1 mM CaCl2, pH 8) to a final enzyme:protein ratio of 1:25 (w/w). Filters were centrifuged (10 min, 14,000 g), rinsed with 50 µL of 50 mM Tris-HCl, pH 8, and centrifuged again (10 min, 14,000 g) to collect digested peptides. To proceed with MS analysis, peptides were purified using C18 spin columns. After activation with 400 µL of acetonitrile, columns were equilibrated twice with 400 µL of water containing 0.1% formic acid (v/v) and then peptides were loaded. After two washing steps with water containing 0.1% formic acid (v/v), digested peptides were eluted twice with 100 μL of 75% ACN in water (v/v) containing 0.1% formic acid (v/v). Purified digested peptides were dried using a stream of nitrogen at room temperature and finally suspended in 100 μL of 95/5 H2O/Acetonitrile (v/v) containing 0.1% formic acid (v/v) and transferred to an autosampler vial for the LC-HRMS/MS analysis. Rat brain protein digests were analyzed by non-targeted proteomics approach based on LC-HRMS/MS and label free quantification. The chromatographic separation was achieved using a reversed phase C18 column (150 × 2.1 mm, 2.6 μm) kept at 30°C. Mobile phases were water (A) and ACN (B), both containing 0.1% formic acid (v/v). The chromatographic gradient was: 0–1 min, 2.5% B; 1–20 min, 2.5–30% B; 20–24 min, 30–50% B; 24–26 min, 50–95% B; 26–30 min, 95% B; 30–30.5 min, 95–2.5% B; 30.5–35 min, 2.5% B. Injection volume was 5 μL and flow rate was 0.2 mL/min. Data were acquired in full-scan data-dependent mode. Full scan spectra were ac-quired at a resolving power of 70,000 full width at half maximum (FWHM) at 200 m/z. Mass range was between 300 and 2000 m/z. Data-dependent fragmentation spectra of the four most intense ions were acquired at a resolution of 17,500 FWHM, using an isolation window of 1.6 m/z, normalized collision energy set to 27, and dynamic exclusion of 30 sec. Source parameters were as follows: sheath gas flow rate was 25 arbitrary units, auxiliary gas flow rate was 10 arbitrary units. Capillary temperature and heater temperature were set to 325°C. The capillary voltage was 3.0 kV in positive polarity electrospray, and the S-lens RF level was 55 V.PrideNot availableMS raw files were analysed using SEQUEST HT as search engine embedded in Proteome Discoverer version 2.1 software. Spectra were searched against the UniProtKB Rattus Norvegicus database (UP000002494) including a decoy database. Trypsin was selected as the enzyme with up to 2 missed cleavages allowed. Carbamidomethyl-cysteine was set as fixed modification, while methionine oxidation and pyro-glutamic conversion at N-terminus were set as variable modification. Peptide and fragment mass tolerances were set at 10 ppm and 0.1 Da, respectively. Principle of maximum parsimony was applied to group proteins into protein families. Percolator algorithm was used to assess confidence of peptide and protein identification, allowing for a maximum false discovery rate of 5%. Only unique peptides were considered for label free relative quantification, which was performed directly by the software by using the precursor area detector function. Quantitative data were examined by principal component analysis (PCA) using the SIMCA-P software (version 13.0). This analysis visualized the distribution of samples based on the proteomic profile of the analysed samples. One-way ANOVA was applied to compare protein abundance of control (C), H2O2-treated (H) and H2O2/MOE (M) samples. Fold changes for the quantified proteins were calculated as the ratio of protein abundance in H₂O₂-treated samples relative to controls (H₂O₂/control), and in H₂O₂-treated samples relative to MOE-treated samples (H₂O₂/MOE). To identify proteins significantly affected by H₂O₂ treatment, we applied selection criteria both based on fold-change and statistical significance. Specifically, we considered as altered proteins with expression values higher than 1.25 or lower than 0.8 (H₂O₂/control) and a p value <0.05. Volcano plot was used to visualize the relationship between fold-change and statistical significance. Among the proteins whose expression was affected by H₂O₂ treatment, we focused on those whose expression was restored after MOE treatment. Indeed, this expression pattern indicates a normalization effect of MOE on protein expression that has been affected by OS.ProteomicsRoberto AngelettiIstituto Zooprofilattico Sperimentale delle Venezie SCS2 - Dept. of Chemistry ItalyPARTIALRattus Norvegicus (rat)42073995 Mattioli LB, Stella R, Peggion C, Cagnin S, Pifferi A, Miraldi E, Cappellucci G, Baini G, Camarda L, Budriesi R, Frosini M. Magnolia officinalis (L.) Bark Extract Counteracts Oxidative Brain Injury: A Proteomic Investigation into Neuroprotective Mechanisms. Int J Mol Sci. 2026 27(8):3350 10.3390/ijms27083350rstella@izsvenezie.itIstituto Zooprofilattico Sperimentale delle VenezieItalyNeurodegenerative diseases involve progressive neuronal loss associated with oxidative stress (OS) and inflammation. Given the limited efficacy of current therapies, natural compounds with multitarget neuroprotective potential are of growing interest. In this study, we investigated the neuroprotective effects of a standardized Magnolia officinalis (L.) bark extract (MOE) in rat brain cortical slices exposed to hydrogen peroxide-induced OS. MOE significantly recovered tissue viability and reduced ROS and malondialdehyde levels caused by OS while attenuating caspase-3, -8, and -9 activation, suggesting modulation of intrinsic and extrinsic apoptotic pathways. Shotgun proteomics using LC-HRMS/MS identified OS-induced protein expression changes reversed by MOE, with fourteen of thirty-three altered proteins rescued by MOE co-treatment. These proteins participate in several processes, including neuronal survival, OS response, and proteostasis. Bioinformatic analysis demonstrated that genes responsible for protein synthesis regulated by MOE are subjected to transcriptional regulation by factors associated with OS, including FOXO4, NRF2, and SP1. The present findings support the hypothesis that MOE exerts multitarget neuroprotective effects by modulating key proteins involved in OS responses and neuronal survival in an acute ex vivo oxidative injury model, suggesting potential relevance for mechanisms associated with NDs.Magnolia officinalis (L.) Bark Extract Counteracts Oxidative Brain Injury: A Proteomic Investigation into Neuroprotective Mechanisms.Mattioli Laura Beatrice LB, Stella Roberto R, Peggion Caterina C, Cagnin Stefano S, Pifferi Alice A, Miraldi Elisabetta E, Cappellucci Giorgio G, Baini Giulia G, Camarda Luca L, Budriesi Roberta R, Frosini Maria MfalseMagnolia officinalis (L.) Bark Extract Counteracts Oxidative Brain In-jury: A Proteomic Investigation into Neuroprotective MechanismsNeurodegenerative diseases involve progressive neuronal loss associated with oxidative stress (OS) and inflammation. Given the limited efficacy of current therapies, natural compounds with multitarget neuroprotective potential are of growing interest. In this study, we investigated the neuroprotective effects of a standardized Magnolia officinalis (L.) bark extract (MOE), rich in honokiol and magnolol (29.1 ± 1.39 mg/g and 16.2 ± 0.81 mg/g, HPLC-UV DAD analysis) in rat brain cortical slices exposed to hydrogen peroxide-induced OS. MOE significantly recovered tissue viability and reduced ROS and malondialdehyde levels caused by OS. Caspase-3, -8, and -9 activation was also attenuated, suggesting a role in modulating both intrinsic and extrinsic apoptotic pathways. Shotgun proteomics using LC-HRMS/MS identified OS-induced protein expression changes reversed by MOE. Thirty-three proteins significantly altered by OS were identified, fourteen of which were rescued by MOE co-treatment. These proteins participate in several processes, including neuronal survival, OS response, and proteostasis. Bioinformatic analysis demonstrated that genes responsible for protein synthesis regulated by MOE are subjected to transcriptional regulation by factors associated with OS, including FOXO4, NRF2, and SP1. The present findings support the hypothesis that MOE exerts multitarget neuroprotective effects by modulating key proteins involved in OS responses and neuronal survival, suggesting a promising natural products-based strategy for pre-venting processes related to neurodegenerative diseases.2026-05-252026-03-16PXD075711NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:309807NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:263737NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:96731NEWT:383379NEWT:10029NEWT:913645NEWT:44491NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:3112NEWT:4442NEWT:192875NEWT:1214915NEWT:12637NEWT:59729NEWT:2164133NEWT:108061NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:1310161NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:5759NEWT:1736231NEWT:5518NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:633149NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:118698NEWT:263NEWT:118696NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:118694NEWT:2850NEWT:118691NEWT:33548NEWT:274NEWT:408172NEWT:408170NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:1436733NEWT:460519NEWT:1247411NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:333760NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:320637NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:261756NEWT:571256NEWT:40483NEWT:272634NEWT:9031NEWT:1872122NEWT:7091NEWT:141262NEWT:108931NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:8694NEWT:4093NEWT:4096NEWT:8692NEWT:185579NEWT:941442NEWT:13076NEWT:1226408NEWT:7108NEWT:40479NEWT:4076NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:4084NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:186441NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:8654NEWT:8658NEWT:1268063NEWT:8655NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:353152NEWT:8496NEWT:27448NEWT:40674NEWT:1194669NEWT:51329NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:104395NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:596153NEWT:229533NEWT:1836NEWT:80863NEWT:105231NEWT:52638NEWT:57075NEWT:4097NEWT:8697NEWT:8695NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:287889NEWT:51515NEWT:588596NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NCBITaxon:714NEWT:632957NEWT:9925NEWT:89462NCBITaxon:9606NEWT:8839NEWT:90690NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:999810NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:231490NEWT:430615NEWT:563041NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:1817808NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:8643NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:8644NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:160235NEWT:72407NEWT:8639NEWT:349741NEWT:409859NEWT:157295NEWT:2096NEWT:192222NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:409860NEWT:2785785NEWT:411901NEWT:8626NEWT:105884NCBITaxon:9615NEWT:8621NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:8624NEWT:8623NEWT:298315NEWT:8618NEWT:70448NEWT:698936NEWT:9940NEWT:3168NEWT:521001NEWT:192011NEWT:1813887NEWT:118702NEWT:118700NEWT:224911NEWT:118705NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:1493NEWT:307972NEWT:1496NEWT:2102NEWT:660469NEWT:45474NEWT:1590841NEWT:4513NE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