Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_2.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_3.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_4.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_1.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_2.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_3.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_4.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_1.mzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_3.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_2.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_4.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_3.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_1.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_sFtsk_2.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_1.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/250501_ZMM_KMI_Ftsk_4.raw.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001492.mztabftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001497.mztabftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001490.mztabftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001495.mztabftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001493.mztabftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001496.mztabftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001491.mztabftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD073126/F001494.mztabprimaryOK200michal.tracz2@uwr.edu.plMichał TraczData-dependent acquisitionMass SpectrometryBottom-up proteomicsChromosome segragationDdaIon mobilityFtskHalo-tagCell divisionSftskMycobacterium smegmatisBacteriaPull-downhttps://www.ebi.ac.uk/pride/archive/projects/PXD073126Cell Suspension CultureLiquid cultures of strains expressing FtsK-HT and sFtsK-HT were cultivated to an optical density (OD600) of approximately 0.8. Biomass was harvested by centrifugation at 5,000 rpm for 20 minutes at 4°C, washed twice with ice-cold PBS, and resuspended in 10 mL of freshly prepared immunoprecipitation (IP) buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.8% Triton X-100) supplemented with a protease inhibitor cocktail (Pierce Protease Inhibitor, Thermo Scientific). Lysis was performed via sonication on ice for a total process time of 25 minutes (50% amplitude; 5-second pulse/5-second pause cycles). The resulting lysate was clarified by centrifugation at 10,000 rpm for 20 minutes at 4°C, followed by a second centrifugation step to ensure complete removal of cellular debris. Total protein concentration was quantified using the Bradford method (ROTI®Quant, Carl Roth). For the affinity purification, 10 mg of total protein lysate was diluted to a final volume of 14 mL with IP buffer. Magne HaloTag beads (100 µl; Promega) were equilibrated by washing three times in IP buffer to remove the storage solution, then added to the lysate and incubated overnight at 4°C with constant rotation. Following incubation, the beads were isolated using a magnetic rack and washed four times with 1 mL of cold IP buffer to remove non-specifically bound contaminants. To remove the detergent from the IP buffer, beads were washed four times in 25 mM Tris pH 7.5. Bead bound proteins were then denatured at 65°C for 10 minutes in 25 mM Tris pH 7.5, 0.1% sodium deoxycholate, 3mM DTT. Subsequently, 200 ng of trypsin was added to the sample for an overnight on-bead digestion in 37°C. Next day the solution was separated from the beads, acidified and sodium deoxycholate was removed by centrifugation. The supernatant was then desalted using a STAGE tip [1]. Obtained peptide pellet was resuspended in 0.1% formic acid (FA), 3% acetonitrile (ACN) solution. LC-MS was performed on an M-Class Acquity UPLC connected to a Synapt XS HDMS equipped with a nanoESI source. Mobile phase A consisted of H2O + 0.1% FA, while mobile phase B of ACN + 0.1% FA. An 8–40% B 40min linear gradient at a 300nL/min flow rate was applied for sample separation on a C18 75μm x 250mm analytical column kept at 45°C. A 5-minute sample trapping step was performed prior to analytical column separation. Data were collected in ESI+ using ion mobility DDA (HDDDA), with MS scan rate of 0.6 seconds, and MS/MS scan rate of 0.2 seconds, both in the 50–2000 m/z range. The top 8 precursors from each MS scan with charges 2+, 3+, and 4+ were selected for MS/MS with 1 scan allowed per transition. Collision energy ramp determined specifically for each m/z (start LM, HM: 20–52 V; end LM, HM: 27–62 V) was applied on the transfer cell. Fragments not matching precursor’s drift time were stripped. Source and IMS conditions were fine-tuned. A (Glu1)-Fibrinopeptide B solution was acquired in-parallel as lockmass, and correction was applied post-acquisition. Four independent biological replicates were analyzed (n=4), 8 samples in total.PrideNot availableRaw processing was performed using PLGS v3.0.3 (Waters). Obtained spectra were exported as .mgf files and searched using Mascot Server v2.8.0.1 (Matrix Science) against the Mycolicibacterium smegmatis protein sequence databank (UP000000757) to which porcine trypsin, human keratins, rat Ig gamma-2A C and Ig lambda-2 C sequences were appended (UniProt entries). Rat Ig sequences were identified in a SwissProt databank pre-search, and are most likely resin related. The search parameters were: peptide mass tolerance: 15 ppm; fragment mass tolerance: 25 ppm; max. protein mass: 1 MDa; digest enzyme: trypsin; max. missed cleavages: 3; variable modification: oxidation of methionine; FDR (PSM): 1%. Protein hits were grouped into families. The Mascot protein hit .csv files were exported for each search, and all families (322) were combined into a single list, along with their score sum, as well as peptide and occurrence counts for either Ftsk or sFtsk, separately. To distinguish proteins specific to FtsK or sFtsK, we assumed hits with no cooccurrence (hit must be present exclusively either in the FtsK pull-down or the sFtsK pull-down), and with a peptide count of at least two (minimal requirement hence being either 1 occurrence by 2 peptides or 2 occurrences with 1 peptide each). This way, 22 and 13 proteins were determined as exclusive for either FtsK or sFtsK, respectively.ProteomicsMichał TraczCOMPLETEMass Spectrometry Laboratory, Faculty of Biotechnology, University of WroclawMycolicibacterium Smegmatis Mkd8michal.tracz2@uwr.edu.pl10.1128/jb.00096-26Laboratory of Mass Spectrometry, Faculty of Biotechnology, University of WroclawPoland10.6019/PXD073126falseInteracome comparision for two FtsK protein variants from Mycobacterium smegmatisThis project aimed at comparing the interaction partners of two FtsK protein variants expressed as HaloTag fusions in Mycobacterium smegmatis. FtsK protein is an important factor in bacterial cell division and chromosomal segregation. The variants were the full length FtsK and an N-terminally shortened FtsK (sFtsk, ~100 aminoacids). HaloTag was C-terminally fused for both variants.2026-05-252026-01-15PXD073126NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:55153NEWT: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: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: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: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: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: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:200302NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:999810NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:430615NEWT: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:6938NCBITaxon:10407NEWT:1736309NEWT:4751NEWT:10360NEWT:47664NEWT:317548NEWT:15819NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:1239NEWT:36745NEWT:272563NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:466585NEWT:1000589NEWT:85963NEWT:85962NE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