Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiP_combined.txt.rarftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/NUP65C_combined.txt.rarftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BIP_BSF_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BioID_PCF_wtcontrol_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BioID_PCF_wtcontrol_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiPN_BSF_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/NUP65C_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BioID_BSF_wtcontrol_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiPN_PCF_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiPN_PCF_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BIP_PCF_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiPN_BSF_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BioID_PCF_wtcontrol_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BIP_BSF_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BioID_BSF_wtcontrol_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BioID_BSF_wtcontrol_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/NUP65C_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiP_PCF_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiPN_PCF_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BIP_PCF_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BiPN_BSF_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/BIP_BSF_3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075517/TriTrypDB-64_TbruceiTREU927_AnnotatedProteins.fastaprimaryOK200zoltnerm@natur.cuni.czMartin ZoltnerData-dependent acquisitionMass SpectrometryTrypanosomiasisTrypanosoma bruceiSecretory pathwayEndoplasmic reticulumNuclear envelopeGolgi apparatushttps://www.ebi.ac.uk/pride/archive/projects/PXD075517Permanent Cell Line CellCell CultureBiP and NUP65 BioID fusion proteins were expressed from the endogenous locus with a TurboID-HA tandem tag fused to the C-terminus of each respective target protein, by modifying one allele via a PCR-based approach reliant on a (modified) version of the pPOT vector system (PMID: 25567099). For the BiP gene (Tb927.11.7510), TurboID-HA was inserted upstream the coding sequence of the BiP C-terminal ER-retention signal-tetrapeptide (MDDL). BiPN was expressed using the pDEX system fusing 2HA-TurboID to BiP residue 415 (PMID: 17512617). A total 5x10^8 cells were used in each replicate. Cells were harvested at 1,400 g, washed once with serum-free medium and pellets rapidly frozen in liquid nitrogen and stored at -80°C. For isolation of biotinylated proteins, each cell pellet was resuspended in 1 ml lysis buffer (0.5% octylphenoxypolyethoxyethanol (IGEPAL), 0.1 M piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES)-NaOH pH 6.9, 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mM MgSO4, 0.1 mM ethylenediaminetetraacetic acid (EDTA), cOmplete EDTA-free protease inhibitor cocktail (Roche)) and incubated for 15 min at room temperature in an orbital mixer. The soluble and non-soluble fractions were separated by centrifugation (14,000 g, 5 min, 4°C) and the soluble fraction incubated with 100 μl streptavidin-linked Dynabeads (MyOne Streptavidin C1, Thermofisher) for 1 hour at 4°C under gentle mixing. Beads were washed twice in 1 ml buffer 1 (2% (w/v) SDS in water) once in 1 ml buffer 2 (0.1% (w/v) deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH7.5, 500 mM NaCl), once in 1ml buffer 3 (250 mM LiCl, 0.5% IGEPAL, 0.5% (w/v) deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and once in 1 ml buffer 4 (50 mM Tris-HCl pH 7.4, 50 mM NaCl); each washing step was 8 minutes at room temperature (RT) under orbital shaking. Beads were then prepared for tryptic digestion by washing three times in 500 µl ice-cold 50 mM NH4HCO3, resuspension in 40 µl of the same buffer supplemented with 10 mM dithiothreitol and incubation in a thermomixer at RT for 1h. Iodoacetamide was added to a concentration of 20 mM, followed by incubation in the dark at RT for 30 min. Finally, 5 μg/ml proteomics-grade trypsin (SOLu-Trypsin, Sigma) and 1 mM biotin was added to the beads. The digest was done overnight at 30°C in a thermomixer (1000 rpm). After removal of the first eluate, beads were resuspended in 50 μl 50 mM NH4HCO3 supplemented with 10 mM dithiothreitol and 5 μg/ml trypsin and incubated in a thermomixer at 37°C for 1h. The eulate was combined with the first eluate and both were dried in a speed-vac. LoBind tubes (Eppendorf) were used throughout. BioID eluted peptides were resuspended in 50 mM NH4HCO3 and passed over C18 stage tip columns as described (PMID: 17703201), then analysed by liquid chromatography-tandem mass spectrometry (LC-MSMS) on an Ultimate3000 nano rapid separation LC system (Dionex) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific).PrideSpectra were processed using the intensity-based label-free quantification (LFQ) in MaxQuant version 2.1.3.0 (PMID: 19029910; PMID: 24942700) searching the T. brucei brucei 927 annotated protein database (release 64) from TriTrypDB (PMID:19843604). False discovery rates (FDR) of 0.01 were calculated at the levels of peptides, proteins and modification sites based on the number of hits against the reversed sequence database. Sample layout: (1) BioID procyclic-form T. brucei with C-terminal TurboID-HA-MDDL fusions for BiP (triplicate): (1.1) BIP_PCF_1.raw, (1.2) BIP_PCF_2.raw, (1.3) BIP_PCF_3.raw (2) BioID bloodstream-form T. brucei with C-terminal TurboID-HA-MDDL fusions for BiP (triplicate): (2.1) BIP_BSF_1.raw, (2.2) BIP_BSF_2.raw, (2.3) BIP_BSF_3.raw (3) BioID procyclic-form T. brucei BiPN-TurboID-HA expression (triplicate): (3.1) BIPN_PCF_1.raw, (3.2) BIPN_PCF_2.raw, (3.3) BIPN_PCF_3.raw (4) BioID bloodstream-form T. brucei BiPN-TurboID-HA expression (triplicate): (4.1) BIPN_BSF_1.raw, (4.2) BIP_BSF_2.raw, (4.3) BIP_BSF_3.raw (5) parental controls boodstream-form T. brucei (triplicate): (5.1) BioID_BSF_wtcontrol_1.raw, (5.2) BioID_BSF_ wtcontrol _2.raw, (5.3) BioID_BSF_ wtcontrol _3.raw) (6) parental controls procyclic-form T. brucei (triplicate): deposited in PXD031245 - (6.1) BioID_PCF_wtcontrol_1.raw, (6.2) BioID_PCF_ wtcontrol _2.raw, (6.3) BioID_PCF_ wtcontrol _3.raw) (7) BioID procyclic-form T. brucei with C-terminal TurboID-HA fusions for NUP65 (duplicate) (7.1) NUP65C_1.raw (7.2) NUP65C_2.rawProteomicsMartin ZoltnerPARTIALHead of Drug Discovery and Evaluation, Charles University in Prague, Department of Parasitology, BIOCEV, Průmyslová 595, 252 50 Vestec, Czech RepublicTrypanosoma Bruceim.zoltner@dundee.ac.ukNot availableDivision of Biological Chemistry & Drug Discovery School of Life Sciences University of Dundee Dundee DD1 5EHUnited KingdomfalseA proximity labelling derived luminal proteome of the Trypanosoma brucei endoplasmic reticulum, Golgi apparatus and nuclear membraneWe employed proximity labelling to derive a proteome of the luminal content of the Trypanosoma brucei endoplasmic reticulum (ER) and Golgi apparatus. We exploited the abundant ER chaperone BiP (Binding-immunoglobulin protein) as ER BioID bait and compare to a truncated version of BiP (termed BiPN) devoid of ER retention and thus trafficked to the Golgi apparatus, as well as parental controls. Our approach covers the two major life cycle stages of T. brucei, i.e., the procyclic form (PCF) and the bloodstream form (BSF). Additionally, we probed the inner nuclear membrane which can be viewed as a specialised ER domain separated by the nuclear pore, via the membrane integral nucleoporin NUP65 as BioID bait in PCF. Altogether, our combined approaches devise an inventory of the T. brucei secretory pathway that complements existing subcellular protein maps derived by proteomics and imaging.2026-04-052026-03-11PXD075517NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:190802NEWT:35554NEWT:150475NEWT:9417NEWT:347515NEWT:1216979NEWT:307972NEWT:544496NEWT:5180NEWT:256737NEWT:115104NEWT:1081927NEWT:67825NEWT:13076NEWT:1249668NEWT:317NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:4081NEWT:876138NEWT:554NEWT:98334NEWT:237561NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:746360NEWT:6239NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:8782NEWT:1000589NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:2261NEWT:4565NEWT:1264690NEWT:6192NEWT:28532NCBITaxon:38727NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:83332NEWT:29760NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:1148NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:7370NEWT:6282NEWT:1134506NEWT:575584NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:1182590NEWT:8726NEWT:10090NEWT:935293NEWT:749200NEWT:4120NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:373995NEWT:544404NEWT:3702NEWT:129249NEWT:8839NEWT:4232NEWT:990119NEWT:4113NEWT:11298NEWT:171101NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:400772NEWT:1097677NEWT:3708NEWT:106592NEWT:9913NEWT:1432138NEWT:10312NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:135588NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:10306NEWT:1911079NEWT:145943NEWT:3635NEWT:5811NEWT:1480154NEWT:235443NEWT:1274414NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:884019NEWT:169963NEWT:36329NEWT:9606NEWT:367830NEWT:157295NEWT:410289NEWT:373153NEWT:915099NEWT:74940NEWT:1450511NEWT:470NEWT:84023NEWT:9838NCBITaxon:9615NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:9940NEWT:573NEWT:9031NEWT:1274420NEWT:70910000-0002-0214-285X