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Sadibolova"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"disease":["Glioblastoma"],"software":[""],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD071225"],"tissue":["Cell Culture"],"sample_protocol":["3D U-87 MG spheroids were lysed with 3% sodium deoxycholate (SDC) in 200 mM triethylammonium bromide (TEAB) and sonicated for 10 min. Lysed samples were then heated at 70 °C for 5 min, sonicated for additional 5 min, treated with benzonase at 37 °C for 30 min, and centrifuged at 4,000 × g for 5 min. Resulting supernatants were transferred to clean tubes and protein concentration was estimated using a microBCA assay (Thermo Fisher Scientific). Fifty µg of protein was reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at 37 °C for 30 min and alkylated with 10 mM S-methyl methanethiosulfonate (MMTS) at 25 °C for 10 min. Proteins were precipitated overnight by adding 5 volumes of prechilled acetone. Samples were centrifuged at 12,000 × g for 5 min and redissolved in 100 mM TEAB. Proteins were digested by a mixture of lysyl endopeptidase and trypsin (Promega) at 1:25 enzyme-to-protein ratio (w/w) at 37 °C overnight. Peptides were labeled with 200 mg of Tandem Mass Tag (TMT) isobaric labels (Thermo Fisher Scientific) (corresponding to the TMT concentration of app. 25 mM) at 25 °C for 60 min. Labeling efficiency and normalization factors within the created multiplex were checked by a 60-min DDA run. Afterwards, the samples were quenched with 50% hydroxylamine (0.2% final concentration) at 25 °C for 15 min and mixed together. Peptides were desalted using Peptide Desalting Spin Columns (Thermo Fisher Scientific) according to the manufacturer’s instructions.   TMT multiplex was redissolved in 2% acetonitrile (ACN)/10 mM ammonium formate (NH4FA). Peptides were separated using XBridge BEH column (C18, 2.5 µm, 2.1 µm × 150 mm) (Waters) using a linear gradient of mobile phase B (80% ACN/10 mM NH4FA) at a flow rate of 0.3 mL/min. The gradient was running from 0% B to 2% B in 2 min, from 2% B to 20% B in 9 min, from 20% B to 50% B in 41 min, and finally from 50% B to 52% B in 5.5 min. The total gradient time was 57.5 min. Fractions were collected into a 96-well polypropylene plate (Agilent Technologies) at 45 s intervals, starting from 3.7 min to 57.7 min, yielding 72 fractions with a volume of 225 µL each. Collected fractions were subsequently concatenated into 24 fractions and evaporated to dryness.  LC-MS/MS analysis was performed on Dionex UltiMate 3000 RSLCnano HPLC system coupled with Orbitrap Exploris 480 mass spectrometer (both Thermo Fisher Scientific) through a NanoSpray Flex (NG) ion source. Concatenated fractions were redissolved in 2% ACN/0.1% trifluoroacetic acid (TFA) and injected onto a PepMap100 C18 trap column (3 µm, 100 Å, 75 µm × 20 mm) and a PepMap RSLC C18 analytical column (2 µm, 100 Å, 75 µm × 250 mm) (both Thermo Fisher Scientific) in two technical replicates. Peptides were firstly loaded onto the trap column at a flow rate of 5 µL/min in 2% ACN/0.1% TFA for 5 min. The separation was achieved using eluent A, consisting of 2% ACN /0.1% formic acid (FA), and eluent B, consisting of 80% ACN/0.1% FA. The gradient ran from 2% B to 34.5% B in 70 min, followed by 34.5% B to 45% B in 10 min and a 5-min washing with 90% B at 250 nL/min. Nanospray ionization source was operated in positive ion mode with spray voltage of 1.8 kV. Full-scan MS spectra were recorded in a profile mode within a scan range of 350-1400 m/z, a resolution of 60,000, an AGC target of 300%, and a maximum injection time of 50 ms. FAIMS Pro Duo interface was operated under compensation voltage of -45V and -60V and at a gas flow rate of 4.6 L/min. Top 10 most intense precursors were isolated using an isolation width of 1.3 m/z, purity threshold of 70%, and a dynamic exclusion of 17 sec, followed by fragmentation with normalized collision energy of 37%. MS2 spectra were recorder with a resolution of 30,000, an AGC target of 200%, and a maximum injection time of 68 ms."],"repository":["Pride"],"modification":[""],"quantification_method":[""],"data_protocol":["Raw files were searched in Proteome Discoverer (version 3.0) using the Sequest HT search engine against the human protein database (UniProt UP000005640, SwissProt and TrEMBL entries, accessed in August 2024). Trypsin/P was used as a cleavage enzyme with up to 2 missed cleavages allowed. Thiomethylation (C) and TMT6plex (K, N-terminus) were set as fixed modifications, while oxidation (M), and N-terminal acetylation, loss of methionine, or their combination were set as dynamic modifications. The precursor and fragment mass tolerances were set to 10 ppm and 0.02 Da, respectively. Target-decoy strategy to control peptide false discovery and identifications were validated by Percolator software. False discovery rate (FDR) was kept at 1%. Only unique peptides assigned to each protein group were considered for quantification. TMT reporter intensities were adjusted using the corresponding isotopic distribution correction factors provided by the manufacturer. Protein intensities were first normalized by equalizing of the total sum of the total reporter ion intensities in each channel. Technical replicates were subsequently averaged.  The analyses were performed using Python 3.9.18 and R 4.3.1. Differential gene expression analysis was performed in R using the limma 3.56.2 package from Bioconductor, which applies linear modeling with empirical Bayes moderation. False Discovery Rate method was used to adjust the p-values.  Gene set enrichment analysis (GSEA) was performed in R using the clusterProfiler 4.8.3 package and in Python using the GSEApy library."],"omics_type":["Proteomics"],"labhead":["Zdenek Hodny"],"instrument_platform":[""],"labhead_affiliation":["Institute of Molecular Genetics of the Czech Academy of Sciences"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["michaela.sadibolova@gmail.com"],"publication":["42222436 Maurencova D, Sadibolova M, Zarska M, Liblova Z, Novak J, Kroupova J, Vasicova P, Bartek J, Hodny Z. Cellular senescence escape and antiviral response discriminate glioblastoma from lower-grade gliomas. Neurooncol Adv. 2026 8(1):vdag122 10.1093/noajnl/vdag122"],"submitter_affiliation":["University Hospital Hradec Kralove"],"submitter_country":["Czechia"],"pubmed_abstract":["<b><i>Background.</i></b> Cellular senescence is a negative prognostic indicator in glioblastoma (GB). However, the specific cell types exhibiting senescence and the molecular mechanisms by which senescent cells (SCs) contribute to GB pathogenesis remain unknown. <b><i>Methods.</i></b> We performed multi-omics integration of publicly available GB patient-derived datasets to identify SCs and their functional impact on GB pathogenesis. We created a transcriptomic definition of SCs to verify their presence in GB datasets. Next, we analyzed transcriptomic profiles of GB and low-grade gliomas to reveal key features of GB aggressiveness. The GB patients' phosphoproteome was analyzed with a focus on key regulators of senescence. To assess chemotherapy-evoked secondary senescence, we performed a proteomic analysis of temozolomide-induced senescence in human GB spheroids. <b><i>Results.</i></b> We identified GB-associated SCs in clusters radial glia, endothelial cells, and immature astrocytes localized primarily in the hypoxic zones. Notably, we identified senescence-escape features and tumor antiviral responses as processes distinguishing GB from low-grade gliomas. In GB samples, we detected inhibitory phosphorylation of p21 and p27 proteins and active PI3K signaling, which can lead to senescence escape and belong to the typical manipulation arsenal of herpesviruses. Our proteomic analysis of temozolomide-induced senescent GB cells reveals that primary (pre-radiochemotherapy) and secondary senescence in GB share similar phenotypic features. Pathways associated with GB aggressiveness are upregulated after therapy, which can promote more aggressive behavior of recurrent tumors. <b><i>Conclusions.</i></b> Our data indicate that senescence and viral reactivation may fuel GB progression, including recurrence, suggesting that senolytics and antiviral drugs are potential therapeutic avenues."],"pubmed_title":["Cellular senescence escape and antiviral response discriminate glioblastoma from lower-grade gliomas."],"pubmed_authors":["Maurencova Dominika D, Sadibolova Michaela M, Zarska Monika M, Liblova Zuzana Z, Novak Josef J, Kroupova Jirina J, Vasicova Pavla P, Bartek Jiri J, Hodny Zdenek Z"],"additional_accession":[]},"is_claimable":false,"name":"Cellular senescence escape and antiviral response discriminate glioblastoma from lower grade gliomas","description":"Cellular senescence is a negative prognostic indicator in glioblastoma (GB). However, the specific cell types exhibiting senescence and the molecular mechanisms by which senescent cells (SCs) contribute to GB pathogenesis remain unknown. We performed multi-omics integration of publicly available GB patient-derived datasets to identify SCs and their functional impact on GB pathogenesis. We created a transcriptomic definition of SCs to verify their presence in GB datasets. Next, we analyzed transcriptomic profiles of GB and low-grade gliomas to reveal key features of GB aggressiveness. The GB patients’ phosphoproteome was analyzed with a focus on key regulators of senescence. To assess chemotherapy-evoked secondary senescence, we performed a proteomic analysis of temozolomide-induced senescence in human GB spheroids. We identified GB-associated SCs in clusters radial glia, endothelial cells, and immature astrocytes localized primarily in the hypoxic zones. Notably, we identified senescence-escape features and tumor antiviral responses as processes distinguishing GB from low-grade gliomas. In GB samples, we detected inhibitory phosphorylation of p21 and p27 proteins and active PI3K signaling, which can lead to senescence escape and belong to the typical manipulation arsenal of herpesviruses. Our proteomic analysis of temozolomide-induced senescent GB cells reveals that primary (pre-radiochemotherapy) and secondary senescence in GB share similar phenotypic features. Pathways associated with GB aggressiveness are upregulated after therapy, which can promote more aggressive behavior of recurrent tumors. Our data indicate that senescence and viral reactivation may fuel GB progression, including recurrence, suggesting that senolytics and antiviral drugs are potential therapeutic avenues.","dates":{"publication":"2026-06-08","submission":"2025-11-25"},"accession":"PXD071225","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2737160","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:57577","NEWT:156563","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:44270","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:93217","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:82569","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:2433","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:1176302","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:77151","NEWT:317447","NEWT:698761","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:1177","NEWT:11308","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","NEWT:314344","NEWT:108061","NEWT:143292","NEWT:224308","NEWT:3134131","NEWT:49928","NEWT:160621","NEWT:110368","NCBITaxon:5204","NEWT:1916073","NEWT:1303873","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:160620","NCBITaxon:77643","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:1410384","NEWT:1310173","NEWT:1072569","NEWT:1072568","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:135666","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1336794","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3710","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:65121","NEWT:3716","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:32201","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:284811","NEWT:2902907","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:10882","NEWT:1431297","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:1423687","NEWT:657322","NEWT:10886","NEWT:3707","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:1301262","NEWT:319224","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:223074","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:28196","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:387344","NEWT:657318","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:422203","NEWT:460523","NEWT:2422","NEWT:2423","NEWT:3755","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:1195466","NEWT:85698","NEWT:291146","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:34839","NEWT:476","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NEWT:106299","NCBITaxon:50557","NEWT:63363","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:427924","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:1120755","NEWT:1862567","NEWT:591090","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:29471","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:546","NEWT:1431340","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:52694","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:40686","NEWT:1225786","NEWT:366497","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:771875","NEWT:377142","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:536","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:480020","NEWT:51315","NEWT:52641","NEWT:353152","NEWT:116597","NEWT:353153","NEWT:116598","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:6055","NEWT:13037","NEWT:518","NEWT:519","NEWT:941638","NEWT:511","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:529120","NEWT:7375","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639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