{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD074929/1_db.proteins.xlsx","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD074929/2_db.proteins.xlsx","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD074929/4_db.proteins.xlsx","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD074929/3_db.proteins.xlsx"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD074929/checksum.txt"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/07/PXD074929/Kraus.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["pavla.postlerova@ibt.cas.cz"],"submitter":["Jan Rasl"],"technology_type":["Mass Spectrometry","Bottom-up proteomics"],"software":["Not available"],"submitter_keywords":["Epididymis","Spermatozoa","Reproduction","Proteomics","Seminal plasma","Protein cargo"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD074929"],"tissue":["Epididymis","Seminal Plasma"],"sample_protocol":["For mass spectrometry analysis, samples containing isolated proteins from EVs were loaded onto a 4–20 % gradient gel (Bio-Rad, Hercules, USA) and were washed in dH2O and stained in 0.05 % (w/v) Coomassie Brilliant Blue R-250 solution (in 35 ml acetic acid; 250 ml ethanol; 215 ml dH2O) at RT overnight. The next day, the gels were incubated in the destaining solution (350 ml ethanol, 100 ml acetic acid, 550 ml dH2O) until the background was decolorized. Each sample lane (EVs from caput, corpus, cauda and SP) was cut into four sections based on molecular weight ranges (200–100 kDa, 100–50 kDa, 50–25 kDa, and 25–10 kDa) (Supplementary Figure S1), placed into 1 % (v/v) acetic acid and then subjected to mass spectrometry (MS) analysis. Firstly, protein bands were excised from SDS-PAGE gels and subjected to destaining in a solution of 25 mM ammonium bicarbonate (AMBIC) and 50 % acetonitrile (ACN) until clear. Following destaining, the gel pieces were dehydrated with 100 % ACN. Proteins were reduced in 100 mM dithiothreitol (DTT) at 60°C for 30 min, then washed sequentially with ultrapure water and ACN. Finally, they were alkylated with 100 mM iodoacetamide (IAA) in the dark for 30 min at 20°C. Additional washes in water and ACN removed excess IAA. To remove N-linked glycans, gel pieces were incubated with PNGase F (New England Biolabs) for 2 hours at 37 °C. After deglycosylation, the gel pieces were washed three times with alternating washes of water and ACN, dried in a SpeedVac, and digested overnight with Trypsin Gold (Promega) in 50 mM AMBIC at 37 °C. The resulting peptides were extracted by incubating the gel pieces with 100% ACN. ACN and the digestion supernatant were combined, dried by SpeedVac, and reconstituted in 0.1% formic acid for subsequent LC-MS/MS analysis. Peptide samples were loaded onto Evotip Pure tips (Evosep) according to the manufacturer’s instructions. Liquid chromatography was performed using the Evosep One system with the standard 30 samples/day method. Peptide separation was achieved on a reverse-phase PepSep C18 analytical column (15 cm × 150 µm, 1.5 µm; Bruker Daltonics). Mass spectrometric analysis was carried out on a timsTOF SCP instrument (Bruker Daltonics) operating in data-dependent acquisition (DDA) mode. The instrument was configured with an ion mobility range of 0.7–1.6 V·s/cm² and a m/z range of 100–1700. The acquisition cycle consisted of a TIMS survey scan (166 ms), followed by five parallel accumulation–serial fragmentation MS/MS scans, resulting in a total cycle time of 1.04 s."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Raw data were analyzed using PEAKS Studio v12.5 (Bioinformatics Solutions Inc.) against the Sus scrofa (Uniprot UP000008227) databases. Variable modifications included methionine oxidation, N-terminal acetylation, and deamidation of asparagine and glutamine residues"],"omics_type":["Proteomics"],"labhead":["Pavla Postlerova"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Laboratory of Reproductive Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czech Republic"],"species":["Sus Scrofa Domesticus (domestic Pig)"],"publication":["42442937 Kraus V, Doleckova B, Frolikova M, Sanovec O, Spevakova D, Pilsova Z, Pilsova A, Komrskova K, Simonik O, Postlerova P. Molecular Profiling of Extracellular Vesicles Isolated from Boar Reproductive Fluids. J Extracell Vesicles. 2026 15(7):e70329 10.1002/jev2.70329"],"submitter_mail":["jan.rasl@ibt.cas.cz"],"submitter_affiliation":["Intitute of Biotechnology"],"submitter_country":["Czechia"],"pubmed_abstract":["Extracellular vesicles (EVs) are small membrane-bound structures that facilitate intercellular communication in the reproductive system, modulating gamete maturation, capacitation, immunomodulation and fertilization. Despite pigs' high relevance as a biomedical model, many aspects of EV biology remain poorly understood. EVs from boar seminal plasma (SP-EVs) are relatively well studied, whereas epididymosomes remain largely uncharacterized. Therefore, this study aimed to isolate and characterize EVs from the caput, corpus and cauda regions of boar epididymis, as well as from SP, with their further precise analysis towards their interaction with sperm. We successfully obtained EVs from all studied fluids with sufficient purity. Importantly, our isolation protocol preserved the EVs' ability to interact with sperm, demonstrated by lipophilic dye staining and biotin labelling experiments, confirming precisely their interaction and cargo transfer to sperm cells. Well-established EV markers, such as Alix and tetraspanins, were detected in the EVs, and additionally, phosphorylated, ubiquitinated and sialylated proteins were uniquely identified. Furthermore, we employed a proteomic approach to characterize EV proteins (mass spectrometry data are available via ProteomeXchange with identifier PXD074929) and investigate their functional roles using the Gene Ontology (GO) database. This study contributes valuable insights into the molecular composition and functional properties of EVs from the male reproductive tract. It may provide a solid framework for further basic and translational research in reproductive biology and biomedicine."],"pubmed_title":["Molecular Profiling of Extracellular Vesicles Isolated from Boar Reproductive Fluids."],"pubmed_authors":["Kraus Veronika V, Doleckova Barbora B, Frolikova Michaela M, Sanovec Ondrej O, Spevakova Daniela D, Pilsova Zuzana Z, Pilsova Aneta A, Komrskova Katerina K, Simonik Ondrej O, Postlerova Pavla P"],"additional_accession":[]},"is_claimable":false,"name":"Molecular profiling of extracellular vesicles isolated from boar reproductive fluids","description":"Extracellular vesicles (EVs) are small membrane-bound structures that facilitate intercellular communication in the reproductive system, modulating gamete maturation, capacitation, immunomodulation, and fertilization. Despite pigs’ high relevance as a biomedical model, many aspects of EV biology remain poorly understood. EVs from boar seminal plasma (SP) are relatively well studied, whereas epididymosomes remain largely uncharacterized. Therefore, this study aimed to isolate and characterize EVs from the caput, corpus, and cauda regions of boar epididymis, as well as from SP, with their further precise analysis towards their interaction with sperm. We successfully obtained EVs from all studied fluids with sufficient purity. Importantly, our isolation protocol preserved the EVs’ ability to interact with sperm, demonstrated by staining with lipophilic dyes and biotin labeling experiments, confirming precisely their interaction and cargo transfer to sperm cells. Well-established EV markers, such as Alix and tetraspanins, were detected in the EVs, and additionally, phosphorylated, ubiquitinated, and sialylated proteins were uniquely identified. Furthermore, we employed a proteomic approach to characterize EV proteins and investigate their functional roles using the Gene Ontology (GO) database. This study contributes valuable insights into the molecular composition and functional properties of EVs from the male reproductive tract. It may provide a solid framework for further basic and translational research in reproductive biology and biomedicine.","dates":{"publication":"2026-07-20","submission":"2026-02-26"},"accession":"PXD074929","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:1073882","NEWT:6819","NEWT:112509","NEWT:475174","NEWT:111205","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:2126337","NEWT:550","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8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