{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/GSCs_differentiatedGSCs_nonGSCs_comparison.xlsx","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/Positive_controls.xlsx"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/checksum.txt"],"Fasta":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/2024_Human_cont_iRT.fasta"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/GSCs_difGSCs_nonGSCs_raw_data.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/Positive_controls_raw_data.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/Positive_controls.sne","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD065565/GSCs_differentiatedGSCs_nonGSCs_comparison.sne"]},"type":"primary"},"statusCodeValue":200,"statusCode":"OK"}],"scores":null,"additional":{"labhead_mail":["helena.skalnikova@lf1.cuni.cz"],"submitter":["Jakub Cervenka"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","diaPASEF","Data-independent acquisition","Bottom-up proteomics"],"disease":["Brain Glioblastoma Multiforme"],"software":[""],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD065565"],"tissue":["Testis","Brain","Primary Cell","Stem Cell","Cell Culture"],"sample_protocol":["In total, 21 samples, including seven GSC cultures and their paired non-GSC and differentiated GSC counterparts, were lysed on ice using RIPA lysis buffer (R0278, Merck) supplemented with protease inhibitors (25 μM pepstatin A, 200 μM AEBSF, and 50 μM E-64). Cell lysates were then processed using our modified filter-aided sample preparation (FASP) protocol, as described in (https://doi.org/10.1111/bpa.13265). Peptides were desalted using Pierce peptide desalting spin columns (Thermo Scientific) and analysed by LC-MS. Peptides (0.5 µg per sample) were first loaded onto a trap column (PepMap Neo C18, 5 mm x 0.3 mm, 5 µm, Thermo Scientific) and subsequently separated on an analytical column (PepSep Ultra C18, 25 cm x 75 µm, 1.5 µm, Bruker Daltonics) using a linear gradient of 2-35% acetonitrile with 0.1% formic acid over 90 min at a flow rate 200 nL/min. The analytical column was maintained at 50°C. Liquid chromatography was performed using a nanoElute 2 system (Bruker Daltonics) directly coupled online to a mass spectrometer timsTOF HT (Bruker Daltonics) equipped with a CaptiveSpray 2 ion source. For data-dependent acquisition (DDA), the default method DDA PASEF-standard_1.1_sec_cycletime (1/K0 range of 0.6-1.6 V·s/cm2, ramp/accumulation time of 100 ms, 10 PASEF ramps per cycle, and a total cycle time 1.17 s) was used in positive ion mode with adjusted MS scan range of 300-1700 m/z. For data-independent acquisition (DIA), the default method dia-PASEF – long gradient (1/K0 range of 0.6-1.6 V·s/cm2, ramp/accumulation time of 100 ms, 16 MS/MS ramps, 32 MS/MS windows, and a cycle time 1.8 s) was used in positive ion mode with adjusted MS scan range of 300-1700 m/z. The two positive control samples (human testis and seminoma, one biological replicate each) were prepared and analyzed in the same way."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["All raw data were analyzed using Spectronaut (19.5.241126.62635, Biognosys). Data-independent acquisition (DIA) data were processed using the directDIA analysis workflow with default BGS Factory Settings. The corresponding data-dependent acquisition (DDA) runs were utilized as library extension files to build a hybrid spectral library. The protein FASTA database was generated by merging the UniProtKB Homo sapiens reference proteome (release 2024_06; updated on 07/10/2024), a list of 381 universal contaminant proteins (https://doi.org/10.1021/acs.jproteome.2c00145), and the iRT sequence (Biognosys). Proteolytic cleavage settings were defined for a combination of Lys-C and trypsin - always cleavage after lysine, and cleavage after arginine – unless proline proceeds. Up to two missed cleavages were permitted. Carbamidomethylation of cysteine was set as a fixed modification, while protein N-terminal acetylation and methionine oxidation were considered variable modifications. Proteins identified by a single stripped peptide sequence (single-hit proteins) were excluded from downstream analysis."],"omics_type":["Proteomics"],"labhead":["Helena Kupcova Skalníkova"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Laboratory of Proteomics, Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague, Czech Republic"],"species":["Homo Sapiens (human)"],"publication":["Not available"],"submitter_mail":["jakub.cervenka@lf1.cuni.cz"],"submitter_affiliation":["Laboratory of Proteomics, Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague, Czech Republic"],"submitter_country":["Czechia"],"additional_accession":[]},"is_claimable":false,"name":"Proteomic analysis of paired glioblastoma stem cells and non-stem cells with focus on PIWI proteins","description":"PIWI proteins, a conserved subfamily of Argonaute proteins that interact with PIWI-interacting RNAs (piRNAs), have been implicated in the pathogenesis of several cancers, including glioblastoma (GBM). This project investigates the expression patterns of the four human PIWI proteins in GBM tissues and glioblastoma stem cells (GSCs) – a therapy-resistant cell population that contributes to tumor aggressiveness and recurrence. We conducted a comprehensive analysis of PIWI expression in GBM. Our results identify PIWIL2 as the predominant PIWI protein significantly enriched in GSCs. Moreover, we report the first evidence of PIWIL4 expression in GBM and question the previously assumed roles of PIWIL1 and PIWIL3 in this context. The deposited data represent mass spectrometry analysis of paired GSCs and non-stem cells derived from human GBMs. In addition, it contains data from GSCs differentiated using 10% fetal calf serum for at least 10 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