Pride

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ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/Pichia_UNIPROT.msfftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS9.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_9.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS7.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_7.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD069498/PK1_BIS11.rawprimaryOK200chiara.dambrosio@cnr.itCHIARA D'AMBROSIOMass SpectrometryGel-based experimentBottom-up proteomicsProbioticsproteomicsmembrane vesiclesimmuno-inflammatory responsespichia kudriavzeviiProbiotics; pichia kudriavzevii; membrane vesicles; proteomics; immuno-inflammatory responseshttps://www.ebi.ac.uk/pride/archive/projects/PXD069498For protein extraction, P. kudriavzevii EVs (2 biological samples) were lysed in parallel with 8 M urea, 1% w/v SDS, containing complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany), at a 1:10 v/v ratio. Cell lysis was initially performed by probe sonication (0.3 cycles) on ice for 2 min, followed by a bath sonication on ice, for an additional 2 min, to enhance lysis efficiency. To ensure complete lysis, the latter step was repeated twice and finally the sample then left on ice for 30 min. Supernatants were collected by centrifugation at 6000 × g for 15 min, at 4 °C, and the corresponding protein concentration was determined with the Pierce BCA Protein assay kit™ (Thermo Fisher Scientific), according to the manufacturer’s instructions. Finally, proteins were precipitated with 6 vol of cold acetone overnight at −20 °C. After precipitation, proteins were pelleted by centrifugation at 8,000 x g for 20 min, at 4 °C, and then vacuum-dried with a SpeedVac roto-evaporator (Thermo Fisher Scientific). Recovered proteins were resolved with preparative 12%T SDS-PAGE under reducing conditions. After protein staining with colloidal Coomassie blue and destaining, whole gel lanes (two in number) were cut into 15 slices, which were separately triturated, washed with water and acetonitrile, treated with 55 mM iodoacetamide in 100 mM NH 4 HCO 3 , washed again, and finally digested with trypsin (12.5 ng/µL) in 50 mM NH 4 HCO 3 . Resulting peptide mixtures were desalted/concentrated using ZipTip μC18 (Millipore, USA), vacuum-dried and finally reconstituted in 5% v/v formic acid. Mass spectrometry Peptide mixtures were analyzed by means of a nanoUltraHighPressure liquid chromatography (nUHPLC)-electrospray (ESI)-Q-Orbitrap-MS/MS platform consisting of a Vanquish-Neo nano- chromatographer (Thermo Fisher Scientific) linked to an Exploris 480 mass spectrometer (Thermo Fisher Scientific) through an easy-spray ion source. Peptides were loaded on an EASY-Spray C18 column (250 mm × 75 μm ID, 2 μm particles, 100 Å pore size) (Thermo Fisher Scientific), and eluted with a gradient of solvent B (19.92/80/0.08 v/v/v water/acetonitrile/formic acid) in solvent A (99.9/0.1 v/v water/formic acid), at a flow rate of 250 nL/min. The gradient of solvent B started at 6%, increased to 31% over 90 min, increased to 50% over 5 min, increased to 95% over 5 min, remained at 95% for 8 min, and finally returned to 6% for equilibrating step. The mass spectrometer operated in data-dependent acquisition mode collecting MS spectra of positive ions within a scan m/z range of 375-1200, using a nominal resolution of 120,000 full width at half maximum (FWHM), an automatic gain control (AGC) target of 3 x 10 6 ions, a maximum injection time (IT) of 50 ms, and a dynamic exclusion value of 45 s, which was followed by MS/MS scans of the 20 most abundant ions. MS/MS spectra were acquired using a normalized collision energy (HCD) of 30%, a resolution of 15,000 FWHM, an AGC target of 2 x 10 5 ions, and a maximum IT of 100 ms.PrideFor protein identification, raw mass data files were merged into Proteome Discoverer v. 3.1 software (Thermo Fisher Scientific, San Jose,USA), enabling database search with Mascot algorithm v. 2.4.2 (Matrix Science, London, UK). Database searching was performed with the following parameters: P. kudriavzevii UniProtKB sequence database (17,479 protein entries), including the most common protein contaminants; carbamidomethylation at Cys as fixed modification; oxidation at Met, deamidation at Asn and Gln, and pyroglutamate formation at Gln as variable modifications. Peptide mass tolerance and fragment mass tolerance were set to ± 10 ppm and ± 0.02 Da, respectively. Trypsin was set as proteolytic enzyme, and the maximum number of missed cleavages was limited to 2. Protein candidates assigned based on at least one sequenced peptide, 2 PSMs and a Mascot score ≥ 25 were considered confidently identified. Definitive peptide assignment was always associated with manual spectra visualization and verification. Results were filtered to 1% false discovery rate. Identified P. kudriavzevii vesicle proteins were associated with components of EVs already assigned in other organisms through a dedicated search in the ExVe database (Parreira et al., 2021). STRING software was used to investigate and visualize the functional relationships among all identified P. kudriavzevii vesicle proteins (Szklarczyk et al., 2023). Functional enrichment analysis, subcellular localization assignment, and further information on biological pathways/molecular processes regarding identified P. kudriavzevii EV proteins were achieved by using dedicated tools assigning diverse Gene Ontology (GO) categories (Aleksander et al., 2023), and COMPARTMENTS (Binder et al., 2014) and Reactome (Milacic et al., 2024) resources available within STRING software.ProteomicsChiara D'AmbrosioPARTIALProteomic, Metabolomic and Mass Spectrometry LAB, ISPAAM, NATIONAL RESEARCH COUNCILPichia Kudriavzevii41901087 Maione A, Matuozzo M, Imparato M, D'Ambrosio C, de Alteriis E, Guida M, Scaloni A, Galdiero E. Extracellular Vesicles from the Probiotic Yeast Pichia kudriavzevii: Proteomic Characterization and Modulation of Immune and Defense Responses in an Induced Inflammation Model of Intestinal Epithelial Cells. Nutrients. 2026 18(6):912 10.3390/nu18060912chiara.dambrosio@cnr.itISPAAM CNRItalyBackground/Objectives: Extracellular vesicles (EVs) derived from probiotics represent a new and exciting frontier in host-microbe therapeutics. These nanoscale carriers are not merely cellular byproducts but are sophisticated mediators of intercellular communication, capable of modulating immune responses, reducing inflammation, and inhibiting pathogens through a rich cargo of bioactive molecules. Methods: The EVs isolated from the culture supernatants of the yeast probiotic candidate Pichia kudriavzevii were characterized for their dimensions, protein composition, and targeting both the gut pathogen virulence and the host inflammatory response. Results: The vesicles had a size distribution from 100 to 150 nm, which is consistent with previous reports on fungal EVs. Proteomic analysis of the purified EVs identified a complex array of 189 proteins, hypothesized to be responsible for some of the antimicrobial and immunomodulatory properties observed. Safety was a key consideration, and the isolated EVs demonstrated no cytotoxicity in human Caco-2 cells and no in vivo toxicity in the Galleria mellonella larval model, confirming their potential for safe use. Conclusions: The field is moving towards a new era of "postbiotics," where cell-free therapies offer a safer, more stable alternative to live probiotics.Extracellular Vesicles from the Probiotic Yeast Pichia kudriavzevii: Proteomic Characterization and Modulation of Immune and Defense Responses in an Induced Inflammation Model of Intestinal Epithelial Cells.Maione Angela A, Matuozzo Monica M, Imparato Marianna M, D'Ambrosio Chiara C, de Alteriis Elisabetta E, Guida Marco M, Scaloni Andrea A, Galdiero Emilia EfalseExtracellular Vesicles from the Probiotic Yeast Pichia kudriavzevii: Proteomic Characterization and Modulation of Immune and Defense Responses in an Induced Inflammation Model of Intestinal Epithelial CellsExtracellular vesicles (EVs) derived from probiotics represent a new and exciting frontier in host-microbe therapeutics. These nanoscale carriers are not merely cellular byproducts but are sophisticated mediators of intercellular communication, capable of modulating immune responses, reducing inflammation, and inhibiting pathogens through a rich cargo of bioactive molecules. The EVs isolated from the culture supernatants of the yeast probiotic candidate Pichia kudriavzevii were characterized for their dimensions, protein composition, and targeting both the gut pathogen virulence and the host inflammatory response. The vesicles had a size distribution from 100 to 150 nm, which is consistent with previous reports on fungal EVs. Proteomic analysis of the purified EVs identified a complex array of 189 proteins, hypothesized to be responsible for some of the antimicrobial and immunomodulatory properties observed. Safety was a key consideration, and the isolated EVs demonstrated no cytotoxicity in human CaCo-2 cells and no in vivo toxicity in the Galleria mellonella larval model, confirming their potential for safe use. The field is moving towards a new era of "postbiotics," where cell-free therapies offer a safer, more stable alternative to live probiotics.2026-04-062025-10-16PXD069498NEWT:330879NEWT:377960NEWT:2042546NEWT:259447NEWT:295546NCBITaxon:1280NEWT:112503NEWT:1129NEWT:309807NEWT:59753NEWT:89184NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:1073882NEWT:6819NEWT:475174NEWT:1117NEWT:498257NEWT:10036NEWT:1590NEWT:10034NEWT:661410NEWT:638632NEWT:224326NEWT:376619NCBITaxon:79857NEWT:1096976NEWT:3765NEWT:1589NEWT:135622NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:656064NEWT:36630NEWT:383379NEWT:418106NCBITaxon:12721NEWT:10029NEWT:913645NEWT:641809NEWT:1392488NEWT:38815NEWT:317447NEWT:4688NEWT:7719NEWT:868565NEWT:135674NEWT:79329NEWT:30069NEWT:12637NEWT:59729NEWT:2164133NEWT:295105NEWT:108061NEWT:60711NEWT:224308NEWT:3347NEWT:160621NEWT:212790NEWT:110368NEWT:1310161NEWT:77133NEWT:145481NEWT:1310165NEWT:29058NCBITaxon:79824NEWT:410658NEWT:44688NEWT:44689NEWT:498211NEWT:347256NEWT:5518NEWT:398007NEWT:1527468NEWT:498217NEWT:498216NEWT:11320NEWT:246196NEWT:246197NEWT:145458NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:3329NEWT:419481NEWT:5507NEWT:410661NEWT:1140NCBITaxon:2157NEWT:1143NEWT:1287689NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:1182590NEWT:3712NEWT:3711NEWT:3714NEWT:2065263NEWT:177437NEWT:10418NEWT:118698NEWT:1616117NEWT:118696NEWT:34865NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:44664NEWT:43346NEWT:3702NEWT:1245466NEWT:3704NEWT:244366NEWT:1246791NEWT:118694NEWT:2850NEWT:118691NEWT:34871NEWT:33548NEWT:52299NEWT:8165NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:460519NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:1348799NEWT:33995NEWT:55784NEWT:1303443NEWT:485NEWT:56636NEWT:2853422NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:1185650NEWT:216778NEWT:1502NEWT:128017NEWT:376686NEWT:95486NEWT:1883446NEWT:1233435NEWT:118223NEWT:109760NEWT:29031NEWT:317047NEWT:491NEWT:460523NEWT:235443NEWT:1757312NEWT:85694NEWT:108458NEWT:5936NEWT:320637NEWT:1078283NEWT:3750NEWT:983964NEWT:11706NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:3747NEWT:1116234NEWT:1255228NEWT:410289NEWT:373153NEWT:1640278NEWT:472NEWT:940825NEWT:529507NEWT:1071661NEWT:470NEWT:5911NEWT:748693NCBITaxon:50557NEWT:39251NEWT:29491NEWT:101841NEWT:446NEWT:153481NEWT:2014887NEWT:33952NEWT:445NEWT:153009NEWT:261756NEWT:166313NEWT:63366NEWT:63367NEWT:215402NEWT:1547NEWT:906914NEWT:9031NEWT:27292NEWT:1050722NEWT:108931NEWT:1293497NEWT:1055524NEWT:150475NEWT:267872NEWT:381666NEWT:172269NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:8694NEWT:33936NEWT:8692NEWT:2903NEWT:185579NEWT:13076NEWT:1006581NEWT:8673NEWT:33940NEWT:550NEWT:554NEWT:451516NEWT:552NEWT:1325291NEWT:36185NEWT:1054211NEWT:1225786NEWT:771870NEWT:51338NEWT:575412NEWT:28112NEWT:6493NEWT:10832NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:643680NEWT:214092NCBITaxon:6157NEWT:13095NEWT:162425NEWT:104105NEWT:216257NEWT:9986NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:9983NEWT:8655NEWT:5147NEWT:28108NEWT:28104NEWT:407821NEWT:568703NCBITaxon:2NEWT:568708NEWT:986NEWT:52641NEWT:28532NEWT:353152NEWT:298176NEWT:227377NEWT:1774284NEWT:40674NEWT:1194669NEWT:443906NEWT:519NEWT:2510939NEWT:7394NEWT:6063NEWT:1328388NEWT:105487NEWT:1548728NEWT:667127NEWT:9557NEWT:377586NEWT:300641NEWT:39655NEWT:9554NEWT:475932NEWT:256318NEWT:206411NCBITaxon:6191NEWT:229533NEWT:214053NEWT:28550NEWT:80863NEWT:90675NEWT:52639NEWT:52638NEWT:57075NEWT:1666905NEWT:8697NEWT:8695NEWT:884204NEWT:1123869NEWT:9544NEWT:9545NEWT:979NEWT:7370NEWT:83906NEWT:99488NEWT:1134506NEWT:255470NEWT:38783NEWT:6426NEWT:33090NEWT:9935NEWT:287889NEWT:305959NEWT:13489NEWT:29829NEWT:92867NEWT:92866NCBITaxon:3055NEWT:51750NEWT:202950NEWT:441772NEWT:48106NEWT:295027NCBITaxon:11320NEWT:632957NEWT:9925NCBITaxon:9606NEWT:90690NEWT:1436183NEWT:300273NEWT:4232NEWT:416348NEWT:11298NEWT:4236NEWT:7742NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:9913NEWT:9915NEWT:105841NEWT:2666255NEWT:999810NCBITaxon:5693NEWT:28995NEWT:2144189NEWT:1392998NEWT:380394NEWT:226900NEWT:1266738NEWT:231490NEWT:244704NEWT:7725NEWT:9909NEWT:9902NEWT:430615NEWT:72664NEWT:563041NEWT:36599NEWT:41949NEWT:249161NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1678078NEWT:749906NEWT:418985NEWT:749907NEWT:150847NEWT:29835NEWT:431947NEWT:69014NEWT:142809NEWT:302409NEWT:130821NEWT:27606NEWT:1519788NEWT:59202NEWT:9975NEWT:8643NEWT:1801631NEWT:1159899NEWT:502780NEWT:860688NEWT:13443NEWT:35231NEWT:8644NEWT:1125630NEWT:5141NEWT:46360NCBITaxon:3044782NEWT:202914NEWT:160235NEWT:502779NEWT:297246NEWT:8639NEWT:1928434NEWT:8637NEWT:1114970NEWT:2096NEWT:285272NEWT:192222NEWT:9963NEWT:1617910NEWT:637379NEWT:12107NEWT:2785785NEWT:411901NEWT:1298722NEWT:8626NEWT:186801NEWT:105884NCBITaxon:9615NEWT:8621NEWT:58334NEWT:58331NEWT:8624NEWT:5116NEWT:8623NEWT:5597NEWT:194439NCBITaxon:12637NEWT:38727NEWT:218496NEWT:8618NEWT:70448NEWT:300251NEWT:9940NEWT:1247190NEWT:118702NEWT:118700NEWT:2126719NEWT:118705NEWT:6945NEWT:2104NEWT:2627626NEWT:6943NEWT:157546NEWT:870726NEWT:91374NEWT:913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