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Reitzel"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["Klebsiella","Pneumoniae","Proteome","Iron.","Exploris","240"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD051800"],"tissue":["Cell Suspension Culture","Macrophage"],"sample_protocol":["Macrophage Infection and Supernatant Preparation  Infections were performed with immortalized BALB/c WT macrophages as previously described (1), with some noted deviations. Macrophage cells were seeded in duplicate on 100 mm treated tissue culture plates at 106 cells per plate in 10 mL Dulbecco’s modified Eagle’s medium (DMEM) (Glutamax DMEM, 10% FBS, 2 mM Glutamax, 1% sodium pyruvate, 1% L-glutamine, 5% pen/strep) for 48 hr at 37°C and 5% CO2. DMEM contains 0.1 mg/L iron in the form of Ferric nitrate (Fe(NO3)3-9H2O ). After 48 hr macrophages achieved confluence (80% well coverage; 107 cells per plate), DMEM media was removed, cells were washed twice with 4 mL room temperature, sterile phosphate buffered saline (PBS), then K. pneuomiae was added to one plate at a multiplicity of infection (MOI) of 100:1 (109 bacteria cells in 10 mL DMEM without antibiotics). The uninfected plate received 10 mL DMEM (without antibiotics) then both plates were incubated at 37°C and 5% CO2 for 90 min. Supernatant was collected and centrifuged at 7,000 RPM for 10 min at 4°C, then syringe filtered using 0.22 µM pore size, under sterile conditions, to remove cells.  Cell Incubation, Lysis, Digestion  From overnight culture, 200 µL of K. pneumoniae was pelleted at 5,000 x g for 10 min at 4°C in biological quadruplicate and technical triplicate. Pelleted cells were washed two times with 1 mL sterile PBS, reconstituted in 200 µL DMEM then added to 2 mL DMEM, uninfected and infected supernatant in quadruplicate and incubated at 37°C for 2 h with 200 RPM shaking. Cells were collected, pelleted at 7,000 RPM for 10 min at 4°C, washed twice with 5 mL sterile PBS resuspended in 300 μL cold 100 mM Tris HCl (pH 8.5) containing protease inhibitor cocktail (Roche). Cells were then lysed, and proteins digested as previously described (1). Briefly, sodium dodecyl sulfate (SDS) was added to 2% final concentration the cells were subjected to probe sonication for 5 cycles at 30% power (30 s on/ 30 s off). Samples were reduced using dithiothreitol (10 mM final concentration; at 95°C for 10 min with 800 RPM shaking) and alkylated at room temperature for 20 min (final concentration 55 mM). Samples were precipitated at -20°C overnight by adding acetone to a final concentration of 80%, followed by centrifuging at 13,200 rpm for 20 min and washing pellet twice with 500 µL 80% acetone.  Pellets were air dried then reconstituted in 100 µL 8 M Urea/40 mM HEPES followed by sonication in ice cold water bath for 15 cycles (30 s on/30 s off). Protein concentration was then determined using BSA tryptophan assay (2) and 50 µg from each replicate of each condition was digested by adding 300 µL 50 mM ammonium bicarbonate and 1 µg LysC/Trypsin (Promega) at room temperature overnight.   LC-MS/MS Analysis Digested samples were dried completely under low pressure in a speed vac at 29°C then reconstituted in 100 µL 0.1% formic acid (FA). Sample concentrations were measured using a nanodrop (NanoDrop One C, Thermo) and 2 µg were loaded onto Evotips according to manufacturer instructions (3). Samples were analyzed using Thermo Scientific Orbitrap Exploris 240 coupled with Evosep One liquid chromatography system using an 88 min method. Mass spectrometry analysis was performed in HCD mode with a 15 cm PepSep column with 150 µm diameter and 1.9 µM beads (Evosep, EV1106). Precursor range was set at 400-2000 m/z, with 60,000 resolution and an intensity threshold of 1e4 and dynamic exclusion was set to 10 sec with charge states 2-8."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["The .RAW files were processed using MaxQuant v2.4.0.0 (4) with default parameters unless stated otherwise. Mass spectra were searched against built-in Andromeda search engine in MaxQuant (5) and resulting peptides were searched against K. penumoniae subsp. pneumoniae K52 serotype FASTA file (5126 protein sequences from UP000000265 proteome ID; accessed Dec. 2, 2022) from Uniprot (6). Trypsin specificity was selected in the digestion conditions, with a maximum of 2 missed cleavages. Cysteine carbamidomethylation was included as a fixed modification and methionine oxidation and N-acetylation of proteins were included as variable modifications. The number of peptides needed to identify a protein was set to 2 with a minimum peptide length of 7 amino acids. Protein abundance was quantified using label-free quantification (LFQ) with ratio count set to 1 (7). Match between runs was enabled with match window of 0.7 min and alignment window of 20 min (7).  Bioinformatics “ProteinGroups.txt” output file from Maxquant was analyzed using Perseus v2.0.7.0 (8). Proteins were filtered and removed if they matched to potential contaminants, matched to the reverse database, or were only identified by site (i.e., in their modified form). Proteins were filtered to retain only those that were present in at least 3 replicates from at least one groups (i.e., valid value filtering), then LFQ intensities were log2 transformed and missing values were replaced using imputation from a normal distribution using 0.3 standard deviation and 1.8 standard deviation down shift. Significant differences in protein abundance were determined by a students t-test using a confidence interval of 95% (i.e., p ≤ 0.05 indicates a significant difference), Benjamini–Hochberg FDR cutoff of 5% and an S0 = 1. One-dimensional (1D) analysis was performed using a Student’s t-test with FDR = 0.05 and S0 = 1 generating a “score” value which represents the relative intensities of proteins identified within a category where 1 indicates a significant enrichment and -1 indicates a significant depletion (9). Proteins were categorized using Gene Ontology (GO) terms (10). ProteoVis was used to create the volcano plot and 1D annotation heat map figures."],"omics_type":["Proteomics"],"labhead":["Jennifer Geddes-McAlister"],"instrument_platform":[""],"labhead_affiliation":["University of Guelph, Guelph, Ontario, Canada. Department of Molecular and Cellular Biology."],"submission_type":["PARTIAL"],"species":["Klebsiella Pneumoniae"],"submitter_mail":["reitzelc@uoguelph.ca"],"publication":["Not available"],"submitter_affiliation":["University of Guelph"],"submitter_country":["Canada"],"additional_accession":[]},"is_claimable":false,"name":"Microfluidic chip-enabled proteome profiling of bacterial infection of macrophages reveals new pathogenic regulators of immune evasion.","description":"Microfluidic chip enabled cell sorting of non-phagocytosed K. pneumoniae cells from BALB/c macrophages during co-culture. These cell populations were analyzed to determine differences in phagocytosed K. pneumoniae versus non-phagocytosed at the protein level. This resulted in the identification of several iron-associated proteins associated with K. pneumoniae that evaded phagocytosis. Evidence in the literature indicated that secreted products from macrophages (i.e., cytokines) significantly increases expression of iron acquisition genes in bacteria, prompting our investigation (and the contents of our submission) into the changes in K. pneumoniae at the proteome-level when exposed to secretion products of macrophages exposed to pathogens (i.e., K. pneumoniae) versus unexposed, with specific emphasis on iron-associated proteins. It was confirmed that pathogen-exposed macrophage supernatant (i.e., activated macrophages induced higher production of iron-associated proteins in exposed K. pneumoniae compared to non-activated macrophages.","dates":{"publication":"2026-03-24","submission":"2024-04-27"},"accession":"PXD051800","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:184922","NEWT:6703","NEWT:3555","NEWT:71647","NEWT:2","NEWT:157546","NEWT:474186","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:1496","NEWT:544496","NEWT:2102","NEWT:2042546","NEWT:259447","NEWT:45351","NEWT:43179","NEWT:4513","NEWT:180454","NEWT:5722","NEWT:1247","NEWT:6938","NEWT:1129","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:309800","NEWT:281395","NEWT:10360","NEWT:1211601","NEWT:876138","NEWT:47664","NEWT:317548","NEWT:3654","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:498019","NEWT:1351","NEWT:1438992","NEWT:1352","NEWT:2649997","NEWT:272563","NEWT:224326","NEWT:1333499","NCBITaxon:79857","NEWT:1096976","NEWT:82688","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:5821","NEWT:1589","NEWT:135622","NCBITaxon:4896","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:28903","NEWT:3880","NEWT:272559","NEWT:28909","NEWT:515849","NEWT:3641","NEWT:383379","NEWT:466585","NEWT:10029","NEWT:913645","NEWT:1000589","NEWT:85963","NEWT:85962","NEWT:143361","NEWT:317447","NEWT:4688","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:398580","NEWT:4442","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:167387","NEWT:84645","NEWT:44586","NEWT:626528","NEWT:3347","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:5888","NEWT:211586","NEWT:747078","NEWT:1282","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:294381","NEWT:197","NEWT:1390363","NEWT:77133","NEWT:288705","NEWT:29176","NEWT:145481","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:556182","NEWT:1270","NEWT:374990","NEWT:1392","NEWT:498217","NEWT:156471","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:156476","NEWT:286","NEWT:391619","NEWT:360104","NEWT:246196","NEWT:287","NEWT:246197","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:161934","NEWT:1148","NEWT:580240","NEWT:5508","NEWT:294128","NEWT:5507","NEWT:11676","NEWT:55571","NEWT:35500","NEWT:1140","NEWT:100226","NEWT:4530","NEWT:1143","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1390","NEWT:1094343","NEWT:1336795","NEWT:296543","NEWT:316435","NEWT:42157","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:3712","NEWT:82380","NEWT:1034304","NEWT:105023","NEWT:866628","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:375146","NEWT:9378","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NEWT:8175","NCBITaxon:1313","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:44544","NEWT:47946","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:1077286","NEWT:243277","NEWT:990119","NEWT:2850","NEWT:408172","NEWT:227321","NEWT:408170","NEWT:493760","NEWT:106590","NEWT:260710","NEWT:257313","NEWT:3827","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:930945","NEWT:106592","NEWT:536231","NEWT:46704","NEWT:1436733","NEWT:460519","NEWT:1187947","NEWT:572307","NEWT:1432138","NEWT:269796","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:1194599","NEWT:54571","NEWT:9598","NEWT:8030","NEWT:1513458","NCBITaxon:40559","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:759272","NEWT:432359","NEWT:46835","NEWT:1182263","NEWT:109757","NEWT:943146","NEWT:2711","NEWT:300852","NEWT:1502","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:508771","NEWT:1883446","NEWT:29159","NEWT:253","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:93061","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:89920","NEWT:29158","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:180923","NEWT:108458","NEWT:272623","NEWT:272624","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:118499","NEWT:169963","NEWT:32644","NEWT:527796","NEWT:225117","NEWT:746128","NEWT:499175","NEWT:109779","NEWT:43665","NEWT:1266464","NEWT:1715989","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:367830","NEWT:1255228","NEWT:178616","NEWT:649908","NEWT:410289","NEWT:373153","NEWT:375451","NEWT:352472","NEWT:357","NEWT:1071661","NEWT:360094","NEWT:470","NEWT:41364","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:559292","NEWT:39491","NCBITaxon:5811","NEWT:29491","NEWT:411464","NEWT:411460","NEWT:2014887","NEWT:2762","NEWT:1174673","NEWT:1328426","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:571256","NCBITaxon:138","NEWT:40483","NEWT:28038","NEWT:1663","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:38293","NEWT:1428","NEWT:327159","NEWT:178876","NEWT:1660","NEWT:327160","NEWT:573","NEWT:9031","NEWT:1872122","NEWT:7091","NEWT:108931","NEWT:241368","NEWT:1055524","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:303","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216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