Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/PeptideRatioResults_CALCULATIONS.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/Ziolkowska_All_Precursors_Initial_Survey.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/Ziolkowska_Triggering-transitions-surequant.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/PeptideRatioResults.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151445ziol_OA_SureQuant_KN12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151443ziol_OA_SureQuant_KN10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151429ziol_OA_SureQuant_M12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151446ziol_OA_SureQuant_KN13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151435ziol_OA_SureQuant_KN2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151440ziol_OA_SureQuant_KN7.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151430ziol_OA_SureQuant_M13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151448ziol_OA_SureQuant_KN15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151434ziol_OA_SureQuant_KN1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151441ziol_OA_SureQuant_KN8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151431ziol_OA_SureQuant_M14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151418ziol_OA_SureQuant_M1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151427ziol_OA_SureQuant_M10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151437ziol_OA_SureQuant_KN4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151442ziol_OA_SureQuant_KN9.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151419ziol_OA_SureQuant_M2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151425ziol_OA_SureQuant_M8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151432ziol_OA_SureQuant_M15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151436ziol_OA_SureQuant_KN3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151428ziol_OA_SureQuant_M11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151444ziol_OA_SureQuant_KN11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151426ziol_OA_SureQuant_M9.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151433ziol_OA_SureQuant_M16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151424ziol_OA_SureQuant_M7.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151447ziol_OA_SureQuant_KN14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151439ziol_OA_SureQuant_KN6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151420ziol_OA_SureQuant_M3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151422ziol_OA_SureQuant_M5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151421ziol_OA_SureQuant_M4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151449ziol_OA_SureQuant_KN16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151423ziol_OA_SureQuant_M6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/602151438ziol_OA_SureQuant_KN5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/20260215_SureQuant_Analysis_02.skylftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/20260215_SureQuant_Analysis_02.sky.viewftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/20260215_SureQuant_Analysis_02.sky.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/20260215_SureQuant_Analysis_02.skyftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD075974/20260215_SureQuant_Analysis_02.skydprimary200OKsylwia.ziolkowska@umed.lodz.plBianka SwiderskaSRM/MRMMass SpectrometryNot availableMitochondriaSurequantPrmOrbitrap astralLeukocytesEvosep onehttps://www.ebi.ac.uk/pride/archive/projects/PXD075974Peripheral Blood Mononuclear CellWhole blood samples were collected into EDTA tubes, aliquoted (200 µL), and stored at −80 °C. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation (30 min, 400 × g, 4 °C) in Gradisol L (AquaMed), washed with PBS, and used for mitochondrial isolation (Mitochondria Isolation Kit for Cultured Cells, Thermo Scientific). Proteins were extracted using RIPA buffer and quantified with the Micro BCA™ Protein Assay on a GloMax®-Multi+ system (Promega). Samples were stored at −80 °C until analysis. Protein digestion was performed in a 96-well format using a modified SP3 protocol. Disulfide bonds were reduced with 10 mM TCEP (1 h, 60 °C) and alkylated with 30 mM CAA (30 min, room temperature, dark). Proteins were captured on Sera-Mag carboxylated magnetic beads (hydrophilic/hydrophobic mix) in 85% acetonitrile with 0.1% formic acid, followed by washing (87% ethanol ×3, 100% acetonitrile ×2). Digestion was carried out overnight at 37 °C using 1 µg trypsin in 100 mM ammonium bicarbonate. Peptides were eluted in MS-grade water and quantified using the Pierce Colorimetric Peptide Assay. For targeted PRM analysis, 11 proteins were selected, with two proteotypic peptides per protein (three for DNLI1 and APEX1), based on PeptideAtlas and UniProt. Corresponding stable isotope-labelled (SIS) peptides (JPT) were used as internal standards. PRM measurements were performed on an Evosep One–Orbitrap Astral platform using SureQuant acquisition. Method optimisation on pooled samples included adjustment of SIS concentrations to achieve MS1 signals of ~1 × 10⁷–1 × 10⁹, selection of precursor charge states (2+, 3+), fragment ions, and trigger thresholds. Analyses were conducted using 1 µg of peptides loaded onto Evotip Pure C18 cartridges with a 30 SPD method (44-min gradient). Full MS scans were acquired at 120 000 resolution (m/z 300–900, AGC 300%, IT 50 ms) with a 7 s cycle time. SureQuant triggering was based on MS1 detection of SIS precursors (±6 ppm). Triggered MS2 scans (150–1700 m/z, 7 500 resolution, AGC 1000%, IT 10 ms, NCE 27%, isolation window 1.0 m/z) required detection of ≥3 fragment ions, followed by high-resolution MS2 of endogenous peptides (60 000 resolution, AGC 1000%, IT 116 ms). Source parameters were set to 2.1 kV (spray voltage) and 275 °C (capillary temperature).PrideNot availableTargeted SureQuant data were processed using Skyline software. Peak integration, manual inspection and correction of chromatographic peaks, and selection of fragment ions for each peptide were performed in Skyline. For most peptides, at least four fragment ions were selected for quantification, with the exception of the peptide HVLDALDPNAYEAFK. One of the three monitored peptides for DNLI1 (DIEQIAEFLEQSVK) was excluded from further analysis due to substantially lower signal quality compared with the remaining peptides. All three peptides selected for APEX1 were retained, while both peptides selected for the remaining proteins were included in the final analysis. Peptide-level quantitative values were exported from Skyline as ratios between endogenous (light) peptide peak areas and the corresponding SIS peptide peak areas (ratio-to-standard). These ratios were subsequently normalised by dividing by the highest observed value for each peptide, resulting in values scaled between 0 and 1. Protein-level abundances were calculated as the geometric mean of the corresponding peptide values. Statistical analyses were performed in Perseus, where descriptive statistics were calculated and group comparisons were carried out using Student’s t-test.ProteomicsSylwia ZiółkowskaPARTIALDepartment of Medical Biochemistry, Medical University of Lodz, 92-215 Lodz, PolandHomo Sapiens (human)42587823 Ziółkowska S, Kosmalski M, Świderska B, Szczypiorowska A, Jarmusz K, Ejsmont M, Wróblewski AM, Szemraj J, Pietras T, Jabłkowska A, Czarny P. Altered Mitochondrial Base Excision Repair and Mitochondrial DNA Instability in Peripheral Leukocytes of Patients with MASLD. Cells. 2026 15(15):1415 10.3390/cells15151415bianka.swiderska@gmail.comMass Spectrometry Laboratory, Institute of Biochemistry and Biophysics Polish Academy of SciencesPolandMetabolic dysfunction-associated steatotic liver disease (MASLD) is a multifactorial metabolic disorder that is strongly associated with mitochondrial dysfunction and oxidative stress, which may potentially compromise the integrity of mitochondrial DNA (mtDNA). However, the role of the base excision repair (BER) pathway-the main mechanism responsible for repairing oxidative lesions in mitochondria-and maintaining mtDNA stability in MASLD remains poorly understood. Here, we analyzed total mRNA expression levels of key BER components in whole-blood samples, along with mitochondrial protein levels of the selected components. Additionally, we assessed the mtDNA copy number and the damage of mtDNA and nuclear DNA in peripheral leukocytes from MASLD patients and healthy controls. We found that MASLD patients differed from controls in mtDNA and nuclear DNA damage, mtDNA copy number, and selected BER-related markers. However, because the MASLD and control groups also differed substantially in age and BMI, these molecular differences should be interpreted as potentially being associated with age- and BMI-related metabolic status rather than attributable to MASLD alone. While several BER-related genes were downregulated at the mRNA level, the corresponding mitochondrial protein levels were not consistently decreased in MASLD (ProteomeXchange: PXD075974), indicating a discordance between transcriptional and protein-level regulation. These results suggest that altered mitochondrial BER and mtDNA instability in peripheral leukocytes may reflect the combined influence of MASLD, aging, obesity, and broader metabolic dysfunction.Altered Mitochondrial Base Excision Repair and Mitochondrial DNA Instability in Peripheral Leukocytes of Patients with MASLD.Ziółkowska Sylwia S, Kosmalski Marcin M, Świderska Bianka B, Szczypiorowska Agnieszka A, Jarmusz Kinga K, Ejsmont Magdalena M, Wróblewski Adam Marek AM, Szemraj Janusz J, Pietras Tadeusz T, Jabłkowska Aleksandra A, Czarny Piotr PfalseImpaired mitochondrial DNA maintenance in NAFLD: associations between mtDNA copy number, mtDNA damage and BER factorsNon-alcoholic fatty liver disease (NAFLD) is characterized by mitochondrial dysfunction and oxidative stress, potentially compromising mitochondrial DNA (mtDNA) integrity. The contribution of base excision repair (BER) to mtDNA maintenance in NAFLD remains poorly understood. Here, we analyzed mtDNA copy number, mtDNA and nuclear DNA damage, and the expression and mitochondrial protein levels of key BER components in leukocytes from NAFLD patients and healthy controls. NAFLD was associated with increased mtDNA and nuclear DNA damage and reduced mtDNA copy number. Gene expression of the BER-initiating glycosylase NEIL1 and downstream factor APEX1 was decreased, whereas LIG1 was upregulated. Proteomic analysis further revealed lower mitochondrial levels of APEX1, PARP1, FEN1, XRCC1, OGG1 and DNLI3 (encoded by LIG3) in NAFLD. MtDNA copy number positively correlated with BER gene expression, while mtDNA damage showed inverse associations. These results highlight a coordinated impairment of mitochondrial BER in NAFLD, linking defective repair to mtDNA instability and mitochondrial dysfunction.2026-08-172026-03-21PXD075974NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2102NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:1247NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:557436NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:79220NEWT: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:4565NEWT:1264690NEWT:515619NEWT:31155NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:3347NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:374990NEWT: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:980415NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:2850NEWT:408172NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:1436733NEWT:460519NEWT:1187947NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:9483NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT: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:2719099NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:317NEWT:1808362NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT: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:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:623NEWT:986NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:198618NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:632NEWT:6183NEWT:6063NEWT:630NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:2925NEWT:29760NEWT:260704NEWT:1719NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NEWT:52638NEWT:4097NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:1329349NEWT:7370NEWT:83906NEWT:607699NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:426688NEWT:208963NEW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