{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/proteinquants_Report.xlsx"],"Tabular":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/SerumExosomes_Report.tsv"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/checksum.txt"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/05_P5_P_R01_Slot1-22_1_1884.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/12_P2_N_R01_Slot1-24_1_1887.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/16_P6_N_R01_Slot1-16_1_1876.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/00_QC-Mix_B1_03_Slot1-10_1_1890.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/09_P9_P_R01_Slot1-26_1_1889.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/02_P2_P_R01_Slot1-21_1_1883.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/13_P3_N_R01_Slot1-20_1_1882.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/17_P7_N_R01_Slot1-19_1_1881.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/11_P1_N_R01_Slot1-14_1_1873.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/Exosomes.sne","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/14_P4_N_R01_Slot1-25_1_1888.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/08_P8_P_R01_Slot1-30_1_1895.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/15_P5_N_R01_Slot1-27_1_1892.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/19_P9_N_R01_Slot1-12_1_1871.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/18_P8_N_R01_Slot1-23_1_1886.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/10_P10_P_R01_Slot1-28_1_1893.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/00_QC-Mix_B1_03_Slot1-10_1_1896.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/06_P6_P_R01_Slot1-15_1_1875.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/20_P10_N_R01_Slot1-29_1_1894.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/00_QC-Mix_B1_02_Slot1-10_1_1879.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/01_P1_P_R01_Slot1-13_1_1872.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/00_QC-Mix_B1_01_Slot1-10_1_1869.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/04_P4_P_R01_Slot1-18_1_1878.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/03_P3_P_R01_Slot1-17_1_1877.d.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD076220/07_P7_P_R01_Slot1-11_1_1870.d.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["evelyne.maes@agresearch.co.nz"],"submitter":["Evelyne Maes"],"technology_type":["Mass Spectrometry","Bottom-up proteomics","Data-independent acquisition"],"disease":["Unusual Infection"],"software":[""],"submitter_keywords":["Mycoplasma bovis infection","Proteomics","Extracellular vesicle","Timstof"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD076220"],"tissue":["Blood Cell","Blood Serum"],"sample_protocol":["Blood (8 mL) was collected in serum collection blood tubes (Vacutainer, BD), inverted 8 times, and left at room temperature for sufficient time to allow for activation of the clotting process. Blood was centrifuged (1,200 × g for 10 mins) and 5 mL of serum was aliquoted and stored at -20 °C until processing for sEV isolation. Serum samples were defrosted at room temperature and clarified by centrifugation for 10 mins at 1,500 × g. The supernatant was further clarified by centrifugation for 10 mins at 10,000 × g. Serum samples were pooled according to farm, infection status (negative or positive), and breed (Friesian/Jersey cross and Friesian). Samples were made up to 10 mL of total serum and if there was not enough serum, samples were topped up to 10 mL using 1× PBS. This resulted in n = 10 samples in each group (M. bovis negative and positive).Small EVs were isolated from pooled serum samples by size exclusion chromatography (SEC) using IZON qEV10 35 nm Gen 2 columns on an automatic fraction collector (AFC) (IZON Science) collecting 5 mL elution fractions.Concentrated EVs were defrosted at room temp and lysed in 200 µL of a freshly prepared lysis buffer (7 M Urea, 2 M Thiourea, 1% Sodium deoxycholate (SDC) in 100 mM Ammonium bicarbonate (AB) buffer), heated at 95 °C for 10 mins, sonicated three times for 5 seconds each with a VC-50 Vibra Cell Ultrasonic Processor, 120V, 50W (Sonics & Materials Inc., Newtown, CT, USA) at an output of 30. Samples were kept on ice between each sonication and, once sonication was completed, centrifuged at 12,000 × g for 10 mins at 4°C. Proteins were precipitated using Methanol/Chloroform precipitation (Wessel & Flügge, 1984) and resuspended in 25 µL of 100mM ammonium bicarbonate buffer and vortexed thoroughly to dissolve the pellet. The protein concentration was measured using a UV-Vis Spectrophotometer (Implen NanoPhotometer) and 14 µg of protein was transferred into a new LoBind tube (Eppendorf) and topped up to 50 µL with 0.1M ammonium bicarbonate buffer. Next, the samples were reduced with 1.25 µL of 200 mM dithiothreitol (DTT) at 56°C for 45 min followed by alkylation by the addition of 4 µL of 200 mM iodoacetamide at room temperature in the dark for 30 min. Proteins were digested into peptides with 1 µg of trypsin/Lys-C mix (1:25 ratio) and incubated overnight in a thermomixer at 37°C. The peptide mixture was desalted using C18 tips according to manufacturer’s instructions and dried in a vacuum concentrator and stored at -20°C until analysed. The dried digests were resuspended in 50 µL 0.1% formic acid prior to injection on the mass spectrometer.The LC-TIMS-MS/MS data acquisition was undertaken using a nanoElute nanoflow ultrahigh pressure LC system (Bruker Daltonics, Bremen, Germany) coupled to the timsTOF Pro2 mass spectrometer (Bruker Daltonics) operating in positive ion mode, using a CaptiveSpray nanoelectrospray ion source (Bruker Daltonics). The source-drying temperature was set to 180°C with a gas flow of 3 L/min and capillary voltage at 1500V. For each of the samples, 200 ng of peptides was loaded on a reversed-phase column (25 cm length, 75 μm inner diameter, 1.6 μm particle size, 120 Å pore size) with a pulled emitter tip (IonOpticks, VIC, Australia). Peptides were separated at 50°C at a flow rate of 300 nL/min using a linear gradient of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in 100% acetonitrile) from 2% to 35% B in 60 min followed by a steep increase to 95% B in 10 min. The column was held at 95% B for 5 min, returned to 2% B over 2 min and then re-equilibrated at 2% B for 13 min resulting in a total runtime of 90 min. The timsTOF Pro 2 was operated in data-independent acquisition Parallel Accumulation–Serial Fragmentation (dia-PASEF) mode. A polygon filter was applied to exclude low m/z, singly charged ions from PASEF precursor selection. The TIMS-MS survey scan was acquired between 0.6–1.6 V s/cm2 and 100–1700 m/z with a ramp time of 166 ms and an acquisition ramp rate of 9.43 Hz. diaPASEF scans were acquired across 32 defined 25 Th isolation windows from 400 to 1200 m/z and a total estimated diaPASEF cycle time of 1.8sec."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["The dia-PASEF files were analyzed using a library-free direct data independent acquisition (DIA) + (Deep) approach in Spectronaut (version 19.3, Biognosys, Switserland). For the database search using the Pulsar search engine, the following settings were used: a swissprot Bos taurus database, combined with Mycoplasma bovis TS01 sequences (downloaded on 26 October 2022, containing 10,558 sequences), Trypsin/P and LysC as enzymes, specific digest type, with up to two missed cleavages allowed; peptide length ranged from 7–52 amino acids; carbamidomethyl of Cysteine as fixed modification, carbamidomethyl (N-term), carbamyl (N-term), carbamylation (KR), deamidation (NQ), oxidation (M) as variable modifications, and 1% false discovery rate (FDR) for peptide-spectrum matches (PSM), peptide, and protein group identification. Mass tolerance of MS and MS/MS was set as dynamic with a correction factor of one."],"omics_type":["Proteomics"],"labhead":["Evelyne Maes"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Bioeconomy Science Institute / AgResearch group, Lincoln, New Zealand"],"species":["Bos Taurus (bovine)"],"submitter_mail":["evelyne.maes@agresearch.co.nz"],"publication":["42221946 Ross MA, Maes E, Lake AVR, Pratt J, Hefer C, Thomas A, Burroughs A, Begg D, Mitchell MD, Roche JR, Heiser A. Inflammation markers are associated with small extracellular vesicle protein signatures in cows with <i>Mycoplasma bovis</i>. Front Vet Sci. 2026 13:1787282 10.3389/fvets.2026.1787282"],"submitter_affiliation":["Agresearch"],"submitter_country":["New Zealand"],"pubmed_abstract":["

Introduction

Mycoplasma bovis (M. bovis) manifests as diverse clinical pathologies in cattle that significantly impacts cow health and productivity worldwide. Current diagnostic methods rely on active infection to detect antibodies and bacterial DNA, however, these methods could be improved for subclinical infections. This is of particular importance in long-term surveillance programs in countries that have decided to eradicate M. bovis, such as New Zealand.

Methods

This study investigated small extracellular vesicles (sEV) from serum samples of M. bovis infected dairy cows to identify protein cargo that may be used as potential diagnostic markers during subclinical infection to complement current testing methods. Small EV were isolated from serum of dairy cows positive for M. bovis infection (n = 45) and their protein cargo compared with those from serum sEV from non-infected dairy cows (n = 49). Proteins were identified using liquid chromatography-ion mobility-tandem mass spectrometry in pooled samples (n = 10/group), resulting in 695 non-redundant top proteins identified across all samples.

Results

Differential protein abundance analysis indicated 90 proteins significantly (q-value < 0.05) different in sEV of infected animals compared with controls. Proteins associated with inflammation and the complement system as well as proteins involved in the oxidative stress pathway, were in greater abundance in sEV from M. bovis positive animals compared with M. bovis negative animals. Several histone proteins and antimicrobial peptides exhibited lower abundance in sEV from M. bovis positive compared with negative cows.

Conclusion

Although this study has not identified a protein candidate specific enough for use as a single diagnostic marker, our results indicate a shift towards a diseased state in M. bovis infected dairy cows and provide valuable insight into sEV biology during M. bovis infection."],"pubmed_title":["Inflammation markers are associated with small extracellular vesicle protein signatures in cows with <i>Mycoplasma bovis</i>."],"pubmed_authors":["Ross Mallory A MA, Maes Evelyne E, Lake Alice V R AVR, Pratt Joel J, Hefer Charles C, Thomas Ancy A, Burroughs Amy A, Begg Douglas D, Mitchell Murray David MD, Roche John R JR, Heiser Axel A"],"additional_accession":[]},"is_claimable":false,"name":"Inflammation markers are associated with small extracellular vesicle protein signatures in cows with Mycoplasma bovisInflammation markers are associated with small extracellular vesicle protein signatures in cows with Mycoplasma bovis","description":"Mycoplasma bovis (M. bovis) manifests as diverse clinical pathologies in cattle that significantly impacts cow health and productivity worldwide. This study investigated small extracellular vesicles (sEV) from M. bovis infected dairy cows to identify protein cargo that may be used as potential diagnostic markers during bacterial latency. Small EV were isolated from serum of dairy cows positive for M. bovis infection (n = 45) and their protein cargo compared with those from serum sEV from non-infected dairy cows (n = 49).","dates":{"publication":"2026-06-08","submission":"2026-03-27"},"accession":"PXD076220","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:44270","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","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:418106","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:317447","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:224308","NEWT:49928","NEWT:160621","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:1230383","NEWT:77133","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:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:135666","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","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:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:1423687","NEWT:657322","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:1301262","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:28196","NEWT:2853422","NEWT:487","NEWT:1679718","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:2423","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","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:476","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:546","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:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:40686","NEWT:1225786","NEWT:771870","NEWT:8665","NEWT:51338","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: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:13037","NEWT:519","NEWT:941638","NEWT:511","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:529120","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:6039","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:280406","NEWT:83906","NEWT:99488","NEWT:99487","NEWT:1134506","NEWT:255470","NEWT:762903","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:266940","NEWT:287889","NEWT:9938","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:483179","NEWT:315750","NEWT:99490","NEWT:632957","NEWT:265619","NEWT:11294","NEWT:754466","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:1479235","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:105812","NEWT:11298","NEWT:4233","NEWT:4236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