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://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD063824/MELAS_Library_5_61.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD063824/MELAS_NHDF_PEO.zipprimaryOK200a.petretto@gmail.comMartina BartolucciMass SpectrometryBottom-up proteomicsmitochondrial diseasesPoydopamine nanoparticles; mitochondrial diseases; oxidative stress; mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; progressive external ophthalmoplegiamitochondrial encephalomyopathy with lactic acidosis and stroke-like episodesPoydopamine nanoparticlesoxidative stressprogressive external ophthalmoplegiahttps://www.ebi.ac.uk/pride/archive/projects/PXD063824FibroblastNHDFs, MELAS-derived and PEO-derived cells were seeded in 25 cm2 tissue culture flasks (Corning®). Upon reaching approximately full confluency, cells were detached through trypsinization, washed in PBS and frozen at -80°C. Pellets were then lysed, reduced, and alkylated in 50 µl LYSE buffer (Preomics) at 95°C for 10 min and sonicated with an Ultrasonic Processor UP200St (Hielscher), 3 cycles of 30 s. To remove the nanoparticles, samples were centrifuged at 20000 g at 4°C for 20 min. Proteins were isolated and digested by the PAC method automated on a KingFisher™ Apex robot (Thermo Fisher Scientific) in a 96-well format as previously described.[28] Briefly, the tip plate was stored in plate #1. Lysate samples were stored in plate #2, in a final concentration of 70% acetonitrile and with magnetic beads in a protein/bead ratio of 1:4 (1:1 SpeedBead Magnetic Carboxylate, 45152105050250 and 65152105050250). Washing solutions were in plates #3–5 (acetonitrile), plate #6 (70% ethanol), and plate #7 (isopropanol). Plate #8 contained 100 µl digestion solution of 25 mM Tris HCl pH 8, LysC (Wako) in an enzyme:protein ratio of 1:100 (w/w), and trypsin (Promega) in an enzyme:protein ratio of 1:50. The protein aggregation was carried out in two steps of 1 min mixing at medium mixing speed, followed by a 10 min pause each. The sequential washes were performed in 2.5 min and slow speed, without releasing the beads from the magnet. The digestion was set to 4 h at 37°C with slow speed. Resulting peptides were analyzed on the Evosep One system using an EASY spray column (150 μm × 15 cm, 2 μm particle size, Thermo Scientific) and the pre-programmed gradient of 30 samples per day, with a flow rate of 0.5 µl/min. The column temperature was maintained at 40°C and interfaced online with the Orbitrap Exploris 480 Mass Spectroscopy (MS, Thermo Scientific) with high-field asymmetric waveform ion mobility spectrometry (FAIMS), Pro Duo Interface (Thermo Scientific). MS analysis was performed in DIA mode. FAIMS CV was set to -45 at standard resolution. Full MS resolution was set to 120000 in a range between 375 and 1500 m/z and with a normalized automatic gain control (AGC) target of 300% with a maximum IT set to Auto. The normalized AGC target value for fragment spectra was set at 1000%. 40 windows of 15 Da were used with an overlap of 1 Da. The resolution was set to 30000 and IT to Auto. The normalized collision energy was set at 30%. All data were acquired in profile mode using positive polarity.PrideNot availableAll data-independent acquisition (DIA) raw files derived from the proteomic analysis were processed with Spectronaut version 18[33] using a library-free approach (directDIA) under default settings. Enzymes / Cleavage Rules were set to Trypsin/P, LysC. The library was generated against the Uniprot Human database (release UP000005640_9606 June 2024). Carbamidomethylation was selected as a fixed modification, while methionine oxidation and N-terminal acetylation were selected as variable modifications. False Discovery rates (FDRs) of peptide-spectrum matches (PSMs) and peptide/protein groups were set to 0.01. For quantification Precursor Filtering was set to Identified (Qvalue), Imputation Strategy was set to None and MS2 was chosen as quantity MS-level. The Protein Quant Pivot Report generated by Spectronaut was statistically evaluated using Perseus software[34] version 1.6.15.0. GO enrichment was obtained with the webserver HumanBase[35] with “skin fibroblast” as background.ProteomicsAndrea PetrettoIRCCS Istituto G GasliniPARTIALHomo Sapiens (human)smartibartolucci@gmail.com10.1021/ACSANM.5C03169IRCCS GasliniItalyfalsePolydopamine nanoparticles as a potential non-pharmaceutical antioxidant tool against mitochondrial disordersMitochondrial disorders are a subset of hereditary diseases characterized by defects in mitochondrial functionality due to mutations in genes in nuclear or mitochondrial DNA. The molecular hallmarks of mitochondrial disorders include a high production of reactive oxygen species (ROS), morphological and functional mitochondrial aberrations, and metabolic impairments. From a systemic perspective, these molecular and cellular impairments can lead to severe disorders affecting the central nervous system, besides to muscles, heart, and gastrointestinal problems. The treatment of mitochondrial diseases with antioxidant moieties like idebenone and resveratrol has been proposed and tested as a potential therapy for these disorders. Polydopamine nanoparticles (PDNPs) are nanostructures derived from the oxidative self-polymerization of dopamine and characterized by high biocompatibility and biodegradability, the ability to be easily functionalized with active molecules, high antioxidant properties, and the ability to act as photothermal conversion agents when irradiated with near infrared (NIR) light. In this work we tested the potential of PDNPs as a non-pharmaceutical treatment for two mitochondrial diseases, namely mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and progressive external ophthalmoplegia (PEO). We tested PDNPs on fibroblasts derived from healthy subjects and fibroblasts derived from MELAS and PEO patients. The cells were first characterized from a molecular point of view through proteomic analysis. Then, the biocompatibility, internalization rate, intracellular localization, and protective antioxidant properties of PDNPs were assessed. Lastly, the antioxidant properties of PDNPs were also confirmed on an in vivo model based on zebrafish embryos. Our data demonstrates the ability of PDNPs to protect both healthy and patient-derived cells from ROS-induced damage, thus preventing the increment of oxidative stress, ROS-induced apoptosis, and mitochondrial dysfunctions. Eventually, PDNPs showed the capacity to protect zebrafish embryos from pro-oxidative stimuli. Altogether, this work demonstrates the potential of polydopamine nanostructures as an effective countermeasure for some of the molecular hallmarks associated with mitochondrial disorders, paving the way for the future exploitation in clinical applications.2026-07-162025-05-10PXD063824NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:1247NEWT:1129NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:172NEWT:296543NEWT:316435NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:2850NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:54571NEWT:9598NEWT:8030NEWT:9483NCBITaxon:40559NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:1055524NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:187878NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:317NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:1276815NEWT:36185NEWT:588858NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:986NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:40559NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:632NEWT:6183NEWT:6063NEWT:630NEWT:49240NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:1719NEWT:703612NEWT:260705NEWT:80863NEW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