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Scott"],"technology_type":["Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["E. coli"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD069957"],"sample_protocol":["Frozen whole bacterial pellets were prepared using the in-StageTip preparation approach. Cells were resuspended in 4% sodium deoxycholate (SDC), 100 mM Tris pH 8.0 and boiled at 95˚C with shaking for 10 minutes to aid solubilisation. Samples were allowed to cool for 10 minutes and then boiled for a further 10 minutes before the protein concentrations were determined by bicinchoninic acid assays (Thermo Fisher Scientific). 100μg of protein for each biological replicate were reduced / alkylated with the addition of Tris-2-carboxyethyl phosphine hydrochloride and iodoacetamide (final concentration 20 mM and 60mM respectively), by incubating in the dark for 1 hour at 45˚C. Following reduction / alkylation samples were digested overnight with Trypsin (1/33 w/w Solu-trypsin, Sigma) at 37˚C with shaking at 1000rpm. Digests were then halted by the addition of isopropanol and TFA (50% and 1% respectively) and samples cleaned up using home-made SDB-RPS StageTips. SDB-RPS StageTips were placed in a Spin96 tip holder 2 to enable batch-based spinning of samples and tips conditioned with 100% acetonitrile; followed by 30% methanol, 1% TFA followed by 90% isopropanol, 1% TFA with each wash spun through the column at 1000 x g for 3 minutes. Acidified isopropanol / peptide mixtures were loaded onto the SDB-RPS columns and spun through, and tips washed with 90% isopropanol, 1% TFA followed by 1% TFA in Milli-Q water. Peptide samples were eluted with 80% acetonitrile, 5% ammonium hydroxide and dried by vacuum centrifugation at room temperature before being stored at -20˚C.  Prepared digested proteome samples were re-suspended in Buffer A* (2% acetonitrile, 0.01% trifluoroacetic acid) and separated using a two-column chromatography setup composed of a PepMap100 C18 20-mm by 75-m trap and a PepMap C18 500-mm by 75-m analytical column (Thermo Fisher Scientific). Samples were concentrated onto the trap column at 5 l/min for 5 min with Buffer A (0.1% formic acid, 2% DMSO) and then infused into a Orbitrap Q-Exactive plus at 300 nl/minute via the analytical columns using a Dionex Ultimate 3000 UPLCs (Thermo Fisher Scientific). 90-minute analytical runs were undertaken by altering the buffer composition from 2% Buffer B (0.1% formic acid, 77.9% acetonitrile, 2% DMSO) to 22% B over 60 min, then from 22% B to 40% B over 10 min, then from 40% B to 80% B over 5 min. The composition was held at 80% B for 5 min, and then dropped to 2% B over 2 min before being held at 2% B for another 8 min. The Q-Exactive plus Mass Spectrometer was operated in a data-dependent mode switching between the collection of a single Orbitrap MS scan (375-1400 m/z and a resolution of 70k) and up to 15 f MS/MS HCD scans of precursors (Stepped NCE 28,30,42%, with a maximal injection time of 50 ms with the Automatic Gain Control set to 5e4 and the resolution to 17.5k)."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Identification and LFQ analysis were accomplished using MaxQuant (v2.4.7.0)4 with datasets searched against the E. coli 11775 (Uniprot: UP000028628) proteome with the addition of the dCAS9 protein sequence to allow the monitoring of silencing induction. Searches were undertaken using a trypsin specificity and allowing for oxidation on Methionine as well as carbamidomethyl on Cysteine as a fix modification. The LFQ and “Match Between Run” options were enabled to allow comparison between samples. The resulting data files were processed using Perseus (v1.4.0.6)5 to compare the growth conditions using student t-tests. For LFQ comparisons biological replicates were grouped and missing values imputed based on the observed total peptide intensities with a range of 0.3σ and a downshift of 1.8σ. To enable comparison between strains, protein groups were mapped to the E. coli K12 reference (Uniprot: UP000000625) using the PATRIC proteome comparison tool."],"omics_type":["Proteomics"],"labhead":["Lachlan J M Coin"],"instrument_platform":[""],"labhead_affiliation":["Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, 792 Elizabeth Street, Melbourne, VIC 3000, Australia."],"submission_type":["PARTIAL"],"species":["Escherichia Coli"],"publication":["42765828 Pitt ME, Zhang J, Nguyen ANT, Hall MB, Jebeli L, Featherstone LA, Myers GSA, Scott NE, Coin LJM. Interrogating the Escherichia coli Epitranscriptome Via CRISPR Interference and Nanopore Native RNA Sequencing. Microbiologyopen. 2026 15(5):e70413 10.1002/mbo3.70413"],"submitter_mail":["nichollas.scott@unimelb.edu.au"],"submitter_affiliation":["University of Melbourne"],"submitter_country":["Australia"],"pubmed_abstract":["Epitranscriptomics has recently gained significant momentum due to technological advances and translational applications; however, studies on bacterial RNA modifications remain limited. Bacterial RNA is notoriously prone to degradation, and methodologies to investigate the epitranscriptome are challenging. Prior research has shown RNA modifications modulate antimicrobial resistance, virulence and pathogenicity. This research employed CRISPR interference to knock down five known Escherichia coli rRNA modification genes (rlmF, rlmJ, rluD, rsmF and rsmG) in three E. coli strains. These isolates were investigated for growth delays, changes in the proteome and the influence on mRNA modifications via native RNA sequencing. CRISPRi adequately silenced the majority of RNA modification genes in E. coli (> 80% reduction). Significant growth delays were associated with rlmF, rluD and rsmF repression. Unique protein pathways corresponding with RNA modification loss were found for rlmJ (TreB, XylF), rluD (CysH, HycB, PutP, TrpB), rsmF (EvgA) and rsmG (OppC). Known rRNA modification sites for rluD (Ψ) and rsmG (m7G) were detected from analysis of nanopore electrical signal; however, only a weak signal was apparent for m6A (rlmF, rlmJ) and m5C (rsmF) modifications. The inhibition of rRNA modifications resulted in mRNA modification changes, including for genes ompC, cspC, dbhA, dbhB and secY. Our work provides an approach for unravelling the epitranscriptome of E. coli to gain insight into its functional role."],"pubmed_title":["Interrogating the Escherichia coli Epitranscriptome Via CRISPR Interference and Nanopore Native RNA Sequencing."],"pubmed_authors":["Pitt Miranda E ME, Zhang Jianshu J, Nguyen An N T ANT, Hall Michael B MB, Jebeli Leila L, Featherstone Leo A LA, Myers Garry S A GSA, Scott Nichollas E NE, Coin Lachlan J M LJM"],"additional_accession":[]},"is_claimable":false,"name":"Proteomic analysis of RNA modification impacts","description":"Proteomic analysis of  E. coli 11775 and the role of RNA modificaiton by silencing RNA modifciaiton with CRISPRi","dates":{"publication":"2026-09-28","submission":"2025-10-27"},"accession":"PXD069957","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","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:57577","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: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:67352","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","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:29064","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:273791","NEWT:3347","NEWT:49928","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:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:33554","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:171719","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","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:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:980415","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:66440","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:34878","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:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","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: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:215402","NEWT:1547","NEWT:1290454","NEWT:906914","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:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:172271","NEWT:1006581","NEWT:8673","NEWT:33940","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:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:595495","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEW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