Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/PhosphoSites.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/Phospho-Analyst_experimental_design.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/proteinGroups.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Control_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Treatment_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Control_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Control_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Control_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Treatment_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Treatment_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Treatment_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Treatment_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Control_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Treatment_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Control_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Control_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Treatment_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Treatment_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Control_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Control_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Control_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_Total_Treatment_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075314/EXP2TL20241112_P24_0789_E2_MaythamH_DDA_FAIMS_ZrIMAC_Treatment_3.rawprimaryOK200terry.lim@monash.eduTerry LimMass SpectrometryTop-down proteomicsphosphoproteomicsneurotoxicityantibiotic toxicodynamics.reverse phase protein arrayapoptosisdna damage signallingPolymyxin b; neurotoxicity; central nervous system; phosphoproteomics; reverse phase protein array; apoptosis; dna damage signalling; antibiotic toxicodynamics.Polymyxin bcentral nervous systemhttps://www.ebi.ac.uk/pride/archive/projects/PXD075314Protein extraction and phosphopeptide enrichment Brain tissue lysates were prepared using a sodium deoxycholate (SDC)-based solubilization protocol. Proteins were reduced, alkylated, and digested with LysC and trypsin. Phosphopeptides were enriched using zirconium ion–immobilized metal affinity chromatography (Zr-IMAC) implemented on a KingFisher automated platform, following established at the Monash proteomics core facilityProteomics and Metabolomics Platform (REF) standard operating procedures. LC-MS/MS acquisition Phosphopeptide-enriched samples were analyzed using a Dionex Ultimate 3000 RSLCnano system coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). Peptides were separated on an Acclaim PepMap RSLC C18 analytical column (75 µm × 50 cm, 2 µm particle size). Data were acquired in data-dependent acquisition mode with FAIMS-assisted gas phase fractionation to enhance phosphosite coverage.PrideNot availableRaw mass spectrometry data were processed using MaxQuant (v2.0.3.1) with the Andromeda search engine. Searches were performed against a reviewed protein database with a 1% false discovery rate applied at both peptide and protein levels. Carbamidomethylation of cysteine was specified as a fixed modification, while oxidation (M), N-terminal acetylation and phosphorylation of serine, threonine and tyrosine were included as variable modifications. Label-free quantification was applied to phosphosites. Across all samples, approximately 11,000 unique phosphosites mapping to ~3,400 proteins were identified, with ~4,000 phosphosites consistently quantified. Differential phosphorylation was assessed using log₂ fold change thresholds ≥ 1 and adjusted p values < 0.05. Unsupervised analyses, including principal component analysis and hierarchical clustering, were used to evaluate global phosphoproteomic structure and condition-specific signalling patterns.ProteomicsTerry LimCentre for Cancer Research, Hudson Institute of Medical Research, Clayton, Australia. Department of Medicine, School of Clinical Sciences, Monash University, Clayton, Australia. 2 Department of Medicine, School of Clinical Sciences, Monash University, Clayton, Australia. 3 Monash Proteomics and Metabolomics Platform, Clinical Proteomics Node, School of Clinical Sciences, Monash University, Clayton, AustraliaRattus Norvegicus (rat)PARTIAL42601573 Hussein M, Ansaf TS, Sian TCCLK, Baker M, Faridi P, Kho ZY, Selvakumar N, Kaye KS, Rao GG, Li J, Velkov T. Systems-Level Phosphoproteomic and RPPA Profiling Reveals Stress and DNA Damage Signalling as Early Drivers of Polymyxin B Neurotoxicity. Mol Neurobiol. 2026 63(1):835 10.1007/s12035-026-06126-xterry.lim@monash.eduMonash UniversityAustraliaPolymyxins remain indispensable last-line antibiotics for multidrug-resistant Gram-negative infections, yet their clinical use in central nervous system (CNS) infections is constrained by poorly understood neurotoxicity. Here, we define the early molecular signalling events underlying polymyxin B-induced CNS toxicity using an integrated phosphoproteomic and Reverse Phase Protein Array (RPPA) approach in rat brain following intracerebroventricular administration. Global phosphoproteomics revealed extensive phosphosite coverage but identified a highly selective set of significantly regulated phosphosites, implicating calcium-dependent signalling, transcriptional stress regulation, synaptic signalling, and cytoskeletal control, while parallel total proteomics showed minimal changes in protein abundance. RPPA profiling independently confirmed coordinated modulation of stress, apoptotic and survival-associated signalling pathways, including p53, CREB, SQSTM1, Bcl-2, and NFκB related nodes. Network and functional enrichment analyses converged on DNA damage signalling, apoptotic regulation and growth factor-mediated pathways as central features of the polymyxin B early neurotoxicity response, while phosphor to total protein analyses demonstrated suppression of proliferative and pro-survival signalling. Together, these data establish phosphorylation-driven signalling reprogramming as a primary early mechanism of polymyxin B-induced neurotoxicity, providing a mechanistic framework that links membrane-active antibiotic exposure to neuronal stress signalling and identifies candidate pathways for toxicity biomarkers and neuroprotective strategies.Systems-Level Phosphoproteomic and RPPA Profiling Reveals Stress and DNA Damage Signalling as Early Drivers of Polymyxin B Neurotoxicity.Hussein Maytham M, Ansaf Thuraya Safaa TS, Sian Terry C C Lim Kam TCCLK, Baker Mark M, Faridi Pouya P, Kho Zhi Ying ZY, Selvakumar Nivedhitha N, Kaye Keith S KS, Rao Gauri G GG, Li Jian J, Velkov Tony TfalseSystems-level phosphoproteomic and RPPA profiling reveals stress and DNA damage signalling as early drivers of polymyxin B neurotoxicityPolymyxins remain indispensable last-line antibiotics for multidrug-resistant Gram-negative infections, yet their clinical use in central nervous system (CNS) infections is constrained by poorly understood neurotoxicity. Here, we define the early molecular signalling events underlying polymyxin B-induced CNS toxicity using an integrated phosphoproteomic and Reverse Phase Protein Array (RPPA) approach in rat brain following intracerebroventricular administration. Global phosphoproteomics revealed extensive phosphosite coverage but identified a highly selective set of significantly regulated phosphosites, implicating calcium-dependent signalling, transcriptional stress regulation, synaptic signalling and cytoskeletal control, while parallel total proteomics showed minimal changes in protein abundance. RPPA profiling independently confirmed coordinated modulation of stress, apoptotic and survival-associated signalling pathways, including p53, CREB, SQSTM1, Bcl-2 and NFκB related nodes. Network and functional enrichment analyses converged on DNA damage signalling, apoptotic regulation and growth factor-mediated pathways as central features of the polymyxin B early neurotoxicity response, while phosphor to total protein analyses demonstrated suppression of proliferative and pro-survival signalling. Together, these data establish phosphorylation-driven signalling reprogramming as a primary early mechanism of polymyxin B-induced neurotoxicity, providing a mechanistic framework that links membrane-active antibiotic exposure to neuronal stress signalling and identifies candidate pathways for toxicity biomarkers and neuroprotective strategies.2026-09-142026-03-06PXD075314NEWT:6945NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:1211601NEWT:876138NEWT:557436NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:79220NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:1000589NEWT:85963NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:286NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:294128NEWT:11676NEWT:55571NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1094343NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:980415NEWT:1603293NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:460519NEWT:1187947NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:1182263NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:3635NEWT:5811NEWT:235443NEWT:272624NEWT:2719099NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:32644NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:357NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1249668NEWT:7108NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:98334NEWT:426428NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:507601NEWT:643680NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NCBITaxon:2NEWT:985076NEWT:1215323NEWT:623NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:198618NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:51329NEWT:243230NEWT:8355NEWT:9685NEWT:7029NEWT:1080772NEWT:1283300NEWT:6183NEWT:6063NEWT:334747NEWT:61235NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:260707NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NEWT:607699NEWT:6282NEWT:426688NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:34073NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:36015NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:242507NEWT:61674NEWT:1117957NEWT:9913NEWT:4100NEWT:1076NEWT:6763NEWT:3498NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:36111NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:1678078NEWT:749906NEWT:418985NEWT:146479NEWT:1911079NEWT:69373NCBITaxon:620NEWT:264203NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:9612NEWT:38865NEWT:51953NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:72407NEWT:349741NEWT:9605NEWT:9606NEWT:157295NEWT:641501NEWT:7668NEWT:915099NEWT:74940NEWT:9721NEWT:137221NEWT:1450511NEWT:1143193NEWT:411901NEWT:9838NEWT:105884NCBITaxon:9615NEWT:9839NEWT:58334NEWT:1193501NEWT:3055NEWT:58331NEWT:6326NEWT:6689NEWT:5476NEWT:299767NEWT:1274432NEWT:1274426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