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The cells were lysed by repeated cycles of sonication on ice 50J under 30% Amplitude. The clear suspension was further incubated at 80˚C for 10 min, spun down at 5000 x g for 10 min at 4˚C. Protein estimation was done using Coomassie Brilliant Blue Plus assay. Tryptic Digestion was done for 50 µg protein from each sample in 40 µl (100 mM TEAB, 0.1% RapiGest). Protein was denatured at 80˚C for 10 min, cooled and reduced in 4 mM DTT at 60°C for 10 min, followed by alkylation (14 mM Iodoacetamide) at RT in the dark for 30 min. IAA was quenched in 7 mM DTT and digestion was done at a 1:50 trypsin:protein ratio at 37˚C for 16 h. The peptide concentration was estimated and 35 µg peptide from each sample were used for TMTPro 18plex labeling as per manufacturer’s recommendations. The peptides in 50 µl with 100 mM TEAB were labelled in a randomized way with respective TMTPro at 1:10 peptide:label ratio and an incubated for 1 hour at 21˚C. Quenched labelling was done with hydroxylamine (0.4% v/v) at RT for 15 min and the samples were pooled equal volume from each channel to multiplex and vacuum dried. The dried samples were re-suspended in 100µM TEAB containing 1 % TFA and subjected to hydrolysis of RapiGest. Then samples were centrifuged for 15 min at 4°C and 13,000 x g to pellet insolubles. The supernatant was desalted using Pierce C18 spin columns as per manufacturer’s instructions. The desalted samples were evaporated under vacuum. The multiplexed TMTPro labelled peptides were fractionated by basic reverse phase chromatography to yield 12 concatenated fractions offline. Fractions were dried under vacuum and resuspended in 0.1% TFA containing 3% acetonitrile.). An equal amount of sample from each fraction was analysed on an Orbitrap Eclipse™ mass spectrometer with an Ultimate 3000 RSLC™ nano system coupled in-line. Peptides were loaded onto the trapping column (Thermo Scientific, PepMap100, C18, 300 μm X 5 mm), using partial loop injection, for three min at a flow rate of 15 μL/min with 0.1% (v/v) FA in 3% acetonitrile and were moved to an analytical column (Easy-Spray C18 75 µm x 500 mm; 2 µm ) at a flow rate of 300 nL/min using a gradient of 97% buffer A (0.1% FA in water) and 3% buffer B (0.1% FA in 80% ACN) for 3 min. Subsequently % of B was raised to 25 % over 140 min, then to 40 % B for additional 13 min. The column was washed at 90% B for 12 min and the percentage of B was then lowered to 3.8 % to allow the column to re-equilibrate for 15 minutes before next injection. Data was acquired in RTS-SPS-MS3 mode using three FAIMS cv’s (-45v, -60v and -75v) and each FAIMS experiment had a max. cycle time of 2s. MS1 scan was carried out at 120,000-resolution (AGC set to 50% (2e5 ions) with a maximum fill time of 30ms) using a mass range of 415-1500 m/z. To avoid repeated selection of peptides for MSMS the program used a 40 sec dynamic exclusion window. MS/MS was performed on the ion trap at turbo scan rate (AGC set to 100% (5e4 ions) with a maximum fill time of 22 ms) with an isolation window of 0.7 m/z and an HCD collision energy of 32 %. Real time search parameters were set as follows: Fasta databases: 1) Parabacteroides merdae, TrEMBL_UP000004276 (accessed on 20230718, containing 4351 seq) or 2) Roseburia_intestinalis, TrEMBL_UP000004828 (accessed on 20220411; containing 4697 seq); with trypsin as the protease, static modifications: cysteine carbamidomethylation (deltaMass=57.0215) and TMTPro (deltaMass=304.207) on lysines and N-terminus, Methionine oxidation was set as variable modification (deltaMass=15.99491). One missed cleavage was allowed, and FDR filtering was enabled. Only 5 peptides per protein were allowed per basic reverse phase fraction (using the close-out option) and maximum search time for RTS was set to 35ms. Ten of the most abundant peptide fragments were selected for SPS-MS3 and MS3 spectrum was acquired in orbitrap at a resolution of 120,000 on the mass range 100-500 m/z with an AGC target of 500% and a maximum injection time of 246ms."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Raw data was imported as fractions and the data analysis was carried out in Proteome Discoverer v3.0. The raw files were submitted to a database search using a predefined workflow using SequestHT and Comet search algorithms against Parabacteroides merdae, UP000004276 (TrEMBL accessed on 20230718, containing 4351 seq) or Roseburia_intestinalis, UP000004828 (TrEMBL, accessed on 20220411, containing 4697 seq). Common contaminant proteins (cRAP fasta from GPM) were added to the database. The spectra identification was performed with the following parameters: MS accuracy, 10 ppm.; MS/MS accuracy, 0.6 Da for spectra acquired in Ion trap mass analyser; missed cleavage, up to 2; fixed modifications, carbamidomethylation of cysteine and TMTpro on lysins and on peptide N-terminus; variable modifications, oxidation of methionine. An intensity-based rescoring of PSMs identified in SequestHT node was performed by Inferys prior to false discovery rate estimation using Percolator. Only rank 1 peptide identifications with high confidence (FDR<1%) were accepted for identification and quantification reporting. Quantification was carried out on the S/N values of reporter ions for the identified peptides. The protein abundance was enumerated by summing the abundances of the connected peptide groups matching to respective proteins. Normalization factor was generated with respect to the summed abundances of all the proteins identified positively to respective protein databases and the normalized abundance values are reported for comparison across datasets. The channel with the highest total abundance is taken as reference and used for correcting the abundance values in rest of the channels with a constant normalization factor per channel."],"omics_type":["Proteomics"],"labhead":["Kiran R Patil"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Medical Research Council Toxicology Unit, University of Cambridge, Cambridge, UK"],"species":["Parabacteroides Merdae","Roseburia Intestinalis"],"publication":["Not available"],"submitter_mail":["sb2487@cam.ac.uk"],"submitter_affiliation":["MRC Toxicology, Uni of Cambridge"],"submitter_country":["United Kingdom"],"additional_accession":[]},"is_claimable":false,"name":"Proteomics for Roseburia intestinalis and Parabacteroides merdae exposed to isosteviol and duloxetine.","description":"The objective of this project was to identify changes in protein expression in R. intestinalis and P. merdae as a response to the treatment with the antidepressant duloxetine, the sweetener isosteviol and a combination of both. 18 samples before and after treatment were collected for each of the two species under anaerobic conditions. The species were inoculated from frozen stock cultures, three replicates each, into mGAM medium and passaged twice over night. Passage two was inoculated into 60 ml (120 ml for R. intestinalis) medium and pre-exposure0 samples were collected directly hereafter (three per species, one sample per replicate). The second pre-exposure samples were collected upon reaching exponential growth, directly prior to splitting each replicate into four equal sized batches, one per condition: Mock (DMSO, 0.2 %), 50 µM duloxetine, 50 µM isosteviol and a combination of both, 50 µM each. This results in 12 treatment samples, three per treatment, whereas each of the replicates can be traced back to one preculture. Bacteria were grown in the presence of the compounds at 37˚C for four hours, before final sample collection. Pre-exposure samples were collected as controls to monitor changes in protein expression in different growth stages.","dates":{"publication":"2026-04-30","submission":"2023-09-06"},"accession":"PXD045140","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","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:592321","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:1599","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","NEWT:1748418","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:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:7719","NEWT:29073","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:2020713","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","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:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","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:3715","NEWT:2065263","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","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:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:1122981","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","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:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","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:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","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:299467","NEWT:9541","NEWT:51350","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:10804","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: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