Pride

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ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD061145/README.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD061145/2024-07-30-decoys-contam-uniprot_UP000000589_mouse_20220419_CrEspC.fasta.fasftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD061145/Yan_set2.7zftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD061145/Yan_gelBand6-15ng_9-10-2024_Slot2-83_14150.d.1.darftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD061145/Yan_gelBand4-15ng_9-10-2024_Slot2-81_14149.d.1.darftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD061145/FragPipe-22.0.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD061145/Yan_gelBand5-15ng_9-11-2024_Slot2-82_14153.d.1.darprimaryOK200foster@msl.ubc.caJenny MoonData-dependent acquisitionMass SpectrometryBottom-up proteomicsEspcColonoidsMucusAir-liquid interfaceEnteric pathogenshttps://www.ebi.ac.uk/pride/archive/projects/PXD061145Region above 225kDa-ladder-band was excised and cut into small pieces. The gel pieces were reduced and alkylated (PMID30259661) and digested with trypsin (New England Biolabs) overnight at 37°C in 50mM ammonium bicarbonate. Fresh trypsin was added next morning for another 3 hours of digestion at 37°C and resulting peptides were extracted and dried on Vacufuge (Eppendorf). The resulting peptides were cleaned on C-18 STop And Go Extraction (STAGE) tips (PMID12498253) using 40% (v/v) acetonitrile and 0.1% (v/v) formic acid in water as the elution buffer. The peptide concentration was estimated using NanoDrop One absorbance at 205nm, scopes method (ThermoFisher). Fifteen nanograms of total peptides were loaded on and separated on-line using NanoElute 2 UHPLC system (Bruker Daltonics) with using Bruker’s PepSep column (25 cm x 75 µm inner diameter, 1.9 µm particle size with 20 µm emitter). The analytical column was heated to 50°C using a column toaster M (Bruker Daltonics). The NanoElute thermostat temperature was set at 7°C. Buffer A consisted of 0.1% formic acid and 0.5% acetonitrile in water, and buffer B consisted of 0.1% formic acid and 0.5% water in acetonitrile. The analysis was performed at 0.30 μL/min flow rate. A 30 min gradient was run from 2% B to 12% B over 15 min, then to 33% B from 15 to 30 min, then to 95% B over 0.5 min, held at 95% B for 7.72 min. Liquid chromatography was coupled to a Trapped Ion Mobility - Time of Flight mass spectrometer with dual TIMS (TimsTOF Pro2; Bruker Daltonics). The Captive Spray ionization source was operated at 1800 V capillary voltage, 3 L/min drying gas, and 180°C drying temperature. During analysis, the TimsTOF Pro 2 was operated with Parallel Accumulation-Serial Fragmentation (PASEF) scan mode for data-dependent acquisition. The MS and MS/MS spectra were collected in positive mode, from m/z 100 Th to m/z 1700 Th, and from ion mobility range (1/ K0) 0.7 V*s/cm2 to 1.35 V*s/cm2. A polygon filter was applied to the mass-to-charge and the ion mobility plane to include the most likely peptide precursors and to reduce singly charged background ions. TIMS-MS scan was set at equal ramp time and accumulation time of 100 ms, at a rate of 9.42 Hz (100% duty cycle). Active exclusion was enabled with a 0.4 min release and reconsidered if their intensity increased more than 4 times. For each TIMS cycle, 5 PASEF MS/MS scans were recorded (total cycle time 0.64 sec). Target Intensity for parent ions was set to 10,000 cts/s with a threshold of 1,000 cts/s. Isolation widths and masses were set starting at 2.07 m/z and 400 m/z, and ending at 3.46 m/z and 1000 m/z, respectively. The collision energy was ramped linearly as a function of mobility value from 27eV at 1/k0 = 0.7 V·s/cm2 to 55eV at 1/k0 = 1.35 V·s/cm2.PrideAll acquired data was searched using FragPipe version 22.0 (PMID: 28394336) against Uniprot’s mouse database (UP000000589). Common contaminants and decoys were added. Mass tolerances were set to ±20 ppm for both precursors and fragment ions. Data was searched with strict trypsin protease specificity with up to 2 missed cleavage. Cysteine carbamidomethylation was set as a fixed modification; N-terminal acetylation, and methionine oxidation were set as variable modifications. MSBooster was enabled along with “Predict RT” and “Predict spectra”. Percolator was selected for PSM validation. Final reports were generated and filtered at 1% FDR at protein level.ProteomicsLeonard J. FosterBiochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, CanadaPARTIALMus Musculus (mouse)40323239 Chen Y, Gilliland A, Liang Q, Han X, Yang H, Chan J, Lévesque D, Moon KM, Daneshgar P, Boisvert FM, Foster L, Zandberg WF, Bergstrom K, Yu HB, Vallance BA. Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus. Gut Microbes. 2025 17(1):2494717 10.1080/19490976.2025.2494717kyungmee@mail.ubc.caUniversity of British ColumbiaCanadaEnteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses remain to be fully defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy appears to involve penetration of the colonic mucus barrier to reach the underlying epithelium. To better define these interactions, we grew colonoids under air-liquid interface (ALI) conditions, producing a thick mucus layer that mimicked in vivo mucus composition and glycosylation. C. rodentium's penetration of ALI-derived mucus was dramatically enhanced upon exposure to sialic acid, in concert with the secretion of two serine protease autotransporter of Enterobacteriaceae (SPATE) proteins, Pic and EspC. Despite Pic being a class II SPATE, and already recognized as a mucinase, it was EspC, a class I SPATE family member, that degraded ALI-derived mucus, despite class I SPATEs not previously shown to possess mucinase activity. Confirming this finding, E. coli DH5α carrying a plasmid that expresses C. rodentium-derived EspC was able to degrade the mucus. Moreover, recombinant EspC alone also displayed mucinolytic activity in a dose-dependent manner. Collectively, our results reveal the utility of ALI-derived mucus in modeling microbe-host interactions at the intestinal mucosal surface, as well as identify EspC as an atypical class I SPATE that shows significant mucinolytic activity toward ALI-derived mucus.Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus.Chen Yan Y, Gilliland Ashley A, Liang Qiaochu Q, Han Xiao X, Yang Hyungjun H, Chan Jocelyn J, Lévesque Dominique D, Moon Kyung-Mee KM, Daneshgar Parandis P, Boisvert François-Michel FM, Foster Leonard L, Zandberg Wesley F WF, Bergstrom Kirk K, Yu Hong B HB, Vallance Bruce A BAfalseDefining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucusEnteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses remain to be fully defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy appears to involve penetration of the colonic mucus barrier to reach the underlying epithelium. To better define these interactions, we grew colonoids under air-liquid interface (ALI) conditions, producing a thick mucus layer that mimicked in vivo mucus composition and glycosylation. C. rodentium’s penetration of ALI-derived mucus was dramatically enhanced upon exposure to sialic acid, in concert with the secretion of two autotransporter (SPATE) proteins, Pic and EspC. Despite Pic being a class II SPATE, and already recognized as a mucinase, it was EspC, a class I SPATE family member, that degraded ALI-derived mucus, despite class I SPATES not previously shown to possess mucinase activity. Confirming this finding, E. coli DH5a carrying a plasmid that expresses C. rodentium-derived EspC was able to degrade the mucus. Moreover, recombinant EspC alone also displayed mucinolytic activity in a dose-dependent manner. Collectively, our results reveal the utility of ALI-derived mucus in modeling microbe-host interactions at the intestinal mucosal surface, as well as identify EspC as an atypical class I SPATE that shows significant mucinolytic activity towards ALI-derived mucus.2026-08-172025-02-24PXD061145NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:55153NEWT:10244NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:96731NEWT:383379NEWT:10029NEWT:913645NEWT:44491NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:3112NEWT:4442NEWT:31155NEWT:192875NEWT:12637NEWT:59729NEWT:2164133NEWT:108061NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:5759NEWT:1736231NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:633149NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:118698NEWT:263NEWT:118696NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:980415NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:118694NEWT:2850NEWT:118691NEWT:33548NEWT:274NEWT:408172NEWT:408170NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:1436733NEWT:460519NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:320637NEWT:2719099NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:261756NEWT:571256NEWT:40483NEWT:272634NEWT:9031NEWT:1872122NEWT:7091NEWT:141262NEWT:108931NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:4093NEWT:4096NEWT:185579NEWT:941442NEWT:13076NEWT:7108NEWT:40479NEWT:4076NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:1268063NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:198618NEWT:353152NEWT:8496NEWT:27448NEWT:1194669NEWT:51329NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:104395NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:596153NEWT:229533NEWT:1836NEWT:2925NEWT:80863NEWT:105231NEWT:52638NEWT:57075NEWT:4097NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:287889NEWT:51515NEWT:588596NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NEWT:632957NEWT:9925NEWT:89462NEWT:8839NEWT:90690NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:200308NEWT:242507NEWT:200302NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:999810NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:430615NEWT:563041NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:1817808NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:160235NEWT:72407NEWT:349741NEWT:157295NEWT:2096NEWT:192222NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:2785785NEWT:411901NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:298315NEWT:70448NEWT:698936NEWT:9940NEWT:3168NEWT:521001NEWT:192011NEWT:1813887NEWT:118702NEWT:118700NEWT:224911NEWT:118705NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:307972NEWT:1496NEWT:2102NEWT:45474NEWT:1590841NEWT:4513NEWT:1247NEWT:6938NEWT:376741NCBITaxon:10407NEWT:1736309NEWT:4751NEWT:10360NEWT:47664NEWT:557436NEWT:317548NEWT:15819NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:1239NEWT:36745NEWT:272563NEWT:79220NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NE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