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BCA assays were performed to quantify the total protein from lysates and 100 μg of protein used for proteomic analysis. SDC solubilized samples were prepared according to the approach of Kulak et al.. Briefly, samples were reduced and alkylated using 20 mM tris(2-carboxyethyl)phosphine / 50 mM Chloroacetamide at 45°C for 30 minutes with shaking at 1500 rpm in the dark (Eppendorf Thermomixer). Trypsin was added in a 1:100 ratio with 1 µg of SOLu-trypsin (Sigma-Aldrich) added to each sample and digestion allowed to proceed for 18 hours at 37°C with shaking at 600 rpm. Trypsin digestion was quenched by the addition of 1.25x volumes of 100% isopropanol and 0.115x volumes of 10% trifluoroacetic acid (TFA) to each sample with the resulting peptides extracted using styrenedivinyl benzene-reverse phase sulfonate (SDB-RPS) (Sigma-Aldrich) StageTips. The resulting SDB-RPS columns were washed with 150 µL 100% acetonitrile (ACN), followed by 150 µL of 30% methanol and 1% TFA by centrifugation at 1000 xg for 5 minutes. Columns were equilibrated with 150 µL of 1% TFA followed by 150 µL of 90% isopropanol and 1% TFA. Samples were loaded onto columns then washed with 150 µL of 90% isopropanol and 1% TFA, followed by 150 µL of 90% ethyl acetate and 1% TFA, then washed with 150 µL of 1% TFA before being eluted with 150 µL of 80% ACN and 5% ammonium hydroxide. Peptides were dried and stored at -20°C till analysis. Four biological replicates were prepared for each group to allow statistical analysis. Reverse phase Liquid chromatography–mass spectrometry: SDB-RPS cleaned peptide samples were resuspended in Buffer A* (2% ACN, 0.1% TFA) and analysed by DIA using a Orbitrap Ascend (Thermo Fisher Scientific) 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 using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific).Samples were concentrated onto the trap column at 5 μL per minute for 5 minutes with Buffer A and then infused into the Ascend at 300 nL/minute via the analytical column. DIA datasets were collected using 125-minute analytical runs by altering the buffer composition from 3% Buffer B to 25% B over 112 minutes, then from 25% B to 40% B over 4 minutes, then from 40% B to 80% B over 1 minute. The composition was held at 80% B for 3 minutes, and then dropped to 3% B over 1 minute before being held at 3% B for another 4 minutes. For DIA, a single MS1 event (120k resolution, AGC 1x106, 350-1100 m/z) and 50 MS2 scans (NCE 30%, 30k resolution, AGC 1x106, 200-2000 m/z and a maximal injection time of 55 ms) of 13.7 m/z in width windows from 360 to 1033.5 m/z were collected.PrideNot availableDIA datasets were searched using DIA-library free analysis in Spectronaut v18.4 utilizing the B. cenocepacia K56-2 and J2315 proteomes as well as the human proteome as above. Oxidation of Methionine was allowed as a variable modification with Carbamidomethyl set as a fixed modification of Cysteine, and the protease specificity set to Trypsin. Protein quantitation was undertaken using the MaxLFQ option in addition to the precursor PEP threshold being altered to 0.01 from the default 0.2 to improve quantitative accuracy, with all single peptide protein matches excluded. Data analysis was undertaken as above utilizing Perseus v1.6.07.ProteomicsNichollas E. ScottPARTIALDepartment of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, AustraliaHomo Sapiens (human)nichollas.scott@unimelb.edu.auNot availableUniversity of MelbourneAustraliafalseOpsonisation improves duel proteomic analysis of Burkholderia cenocepacia infections. -DIA infection +/- Opsonisation of T6SS and TecAThe opportunistic Cystic Fibrosis (CF) pathogen Burkholderia cenocepacia is associated with severe lung infections. Within the CF lung, macrophages are both a crucial reservoir and a key mediator of the intense inflammatory response characteristic of B. cenocepacia infections. Despite the importance of macrophages for B. cenocepacia pathogenesis, there is a paucity of insight into how B. cenocepacia internalisation and replication impacts both the host and B. cenocepacia proteomes. A key limitation for understanding the proteomic changes during intracellular replication of B. cenocepacia has been the low infectivity of this pathogen, resulting in the generation of mixed cell populations dominated by uninfected cells within in vitro models. Using antibody-mediated opsonization, we show that by improving the efficiency and uniformity of B. cenocepacia internalization this enhances dual proteomic analysis. Comparing opsonized and non-opsonized infections, we show that changes in both host and internalized B. cenocepacia can be assessed in a single experimental framework. At the proteome level, opsonization dramatically improves the detection of protein changes, enhancing the detection of pro-inflammatory signaling and macrophage activation markers. Utilizing this dual proteomic approach, we assess the impact of the B. cenocepacia type 6 secretion system (T6SS) and the T6SS effector TecA at 3 and 24 hours post infection demonstrating that the presence/activities of the T6SS or TecA do not greatly modulate the proinflammatory response of THP-1 cells. Thus, this work demonstrates a simple means for enhancing proteomic analysis of B. cenocepacia infections enabling dual proteomic studies.2026-05-132026-01-18PXD073205NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:71647NEWT:2NEWT:157546NEWT:474186NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:1496NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:4513NEWT:180454NEWT:5722NEWT:1247NEWT:6938NEWT:1129NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:44139NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NEWT:2164133NCBITaxon:183674NEWT:1316931NEWT:224308NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:511145NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:431241NEWT:931281NEWT:4550NEWT:1000561NEWT:2133NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NEWT:145481NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:246196NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:161934NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:5507NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:644042NEWT:296543NEWT:316435NEWT:42157NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:1034304NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:9378NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:1077286NEWT:243277NEWT:990119NEWT:7067NEWT:2850NEWT:1111708NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:930945NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:1194599NEWT:272844NEWT:54571NEWT:9598NEWT:8030NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:537457NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:508771NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:29158NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:1715989NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:1679NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NCBITaxon:138NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:1055524NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:187878NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:40479NEWT:317NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:273068NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:36185NEWT:588858NEWT:1225786NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:197221NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:434271NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:89764NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:38018NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:2059687NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:986NEWT:52641NEWT:7038NEWT:7159NEWT:6192NEWT:28532NCB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