Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap-all.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap32.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap23.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap27.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap28.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap19.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap31.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap01.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap06.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap05.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap09.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap22.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap00.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap26.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap18.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap08.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap30.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap21.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap04.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap17.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap25.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap07.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap20.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap03.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap29.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap02.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/10/PXD065121/Hammond_Ppap24.rawprimary200OKJeremy.Wideman@asu.eduYu-Ping PohMass SpectrometryBottom-up proteomicsSubcellular proteomicsDiplonemidhttps://www.ebi.ac.uk/pride/archive/projects/PXD065121Cell CultureStrain, culture conditions and preparation for lysis Paradiplonema papillatum (ATCC50162) cells were cultivated axenically in Diplo media (36 g/L sea salts [Sigma], with 1g Tryptone and 10ml Foetal Bovine Serum [Sigma], 0.22 m filter sterilised) at 22°C. Cell cultures were harvested in a combined volume of 750ml per sample, harvested at ~2.5E6 cells/ml, processed in triplicates. Cell cultures were concentrated by centrifugation (900xg for 10 minutes) and pellets were resuspended in 6ml detergent free lysis buffer (0.25 M sucrose, 10 mM HEPES, pH 7.4 2mM EDTA, 2mM Mg(OAc) with HaltTM Protease and Phosphatase Inhibitor Cocktail, pre-chilled to 4°C. Cell lysis and fractionation Cell suspension underwent lysis via nitrogen cavitation at 250 psi for 10 minutes (Parr 4639, Parr Instrument Co.). Cell lysate was gently released from chambre to minimise foaming, with collected sample undergoing differential centrifugation following previously established protocol (Geladaki, Britovšek et al. 2019). Briefly, cell lysate underwent centrifugation at speeds described in Table 1, with supernatant being transferred to fresh 2ml centrifuge tubes and undergoing subsequent centrifuge steps, with pellets from previous spins stored at -80°C after collection, in addition to the supernatant fraction from final spin. Pelleted cell lysate was additionally collected and stored for proteomic analysis. Fractional assessment To assess distribution and enrichment of proteins across P. papillatum fractions, immunoblotting was performed using antibodies against ATP synthase subunit (kindly provided by A. Zíková)(Šubrtová, Panicucci et al. 2015), Grp75 (Enzo) (Joseph, Adhihetty et al. 2013)and Grp78 (Novus) (Chou, Yang et al. 2020). Pellets were resuspended in 2x Laemmli buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.004% Bromophenol blue) without DTT. Fractional samples were quantified using the Pierce™ BCA Protein Assay (Thermo Scientific). 10 µg of protein was loaded onto an SDS-PAGE gel (Invitrogen™ Bolt™ Bis-Tris Plus Mini Protein Gels, 4-12%, 1.0 mm, WedgeWell™ format) along with a protein marker (Amersham™ ECL™ Rainbow™ Marker - Full Range). The gel was run for 1 hour at 130 V, briefly washed in 1X PBS buffer, and transferred onto a methanol-activated PVDF membrane (iBlot™ 2 Transfer Stacks, PVDF, Invitrogen™) using the iBlot™ 2 Western Blot Transfer Device (Invitrogen™). The membrane was blocked in 5% non-fat dry milk and 1X PBS buffer for 1 hour at room temperature, followed by incubation with relevant antibodies diluted (1:10000 for ATP-β, and 1:1000 for Grp75 and Grp78) in blocking solution (1% non-fat dry milk in 1X PBS buffer). Blots were incubated at room temperature for 1 hour, then overnight at 4°C. The following day, blots were washed three times for 10 minutes each in 1X PBS, probed with HRP-linked secondary antibodies (31460, Invitrogen) diluted 1:1000 in blocking solution for 1 hour at room temperature, and rinsed again three times for 10 minutes each in 1X PBS-T. Detection was performed using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific™), and imaging was conducted with the Azure 600 (Biosystems). Sample preparation and LC-MS Analysis Native protein pellets obtained from differential centrifugation were digested and desalted following the protocol for the S-Trap™ Micro Column (ProtiFi, USA). Protein concentration was quantified using the BCA assay (Thermo Scientific, USA), while peptide concentration was measured using a fluorometric kit (Thermo Scientific, USA).PrideNot availableLiquid-chromatography tandem mass spectrometry LC-MS/MS analyses of P. papillatum peptides were performed at the Biosciences Mass Spectrometry Core Facility, Arizona State University. Data-dependent mass spectra were collected in positive mode using an Orbitrap Fusion Lumos mass spectrometer coupled with an UltiMate 3000 UHPLC (Thermo Scientific). Peptides were fractionated on an Easy-Spray LC column (50 cm × 75 μm ID, PepMap C18, 2 μm, 100 Å) with an upstream trap column. Each sample was analyzed in technical triplicate. LC-MS settings: electrospray potential 1.6 kV, ion transfer tube temperature 300°C, and the “Universal” peptide analysis method. Full MS scans (375–1500 m/z) were acquired at a resolution of 120,000 with 3-second cycle times. The RF lens was set to 30%, AGC to “Standard,” and monoisotopic peak determination included charge states 2–7. Dynamic exclusion was 60 seconds with a 10 ppm mass tolerance. MS/MS spectra were acquired in centroid mode with a quadrupole isolation window of 1.6 m/z and CID energy of 35%. Peptides were eluted over a 240-minute gradient at 0.25 µL/min using 2–80% acetonitrile/water: 0–3 min (2%), 3–75 min (2–15%), 75–180 min (15–30%), 180–220 min (30–35%), 220–225 min (35–80%), 225–240 min (80–5%). LC-MS/MS analysis of the digested peptides were performed on an EASY-nLC 1200 (Thermo Fisher Scientific) coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Peptides were separated on an Aurora UHPLC column (25 cm × 75 µm, 1.6 µm C18, AUR2-25075C18A, Ion Opticks) with a flow rate of 0.35 µL/min for a total duration of 135 min ionized at 1.6 kV in the positive ion mode. The gradient was composed of 2% solvent B (5 min), 2-6% B (7.5 min), 6-25% B (82.5 min), 25–40% B (30 min), 40-98% B (1min) and 98% B (15min); solvent A: 2% ACN and 0.2% FA in water; solvent B: 80% ACN and 0.2% FA. MS1 scans were acquired at the resolution of 120,000 from 350 to 1,600 m/z, AGC target 1e6, and maximum injection time 50 ms. MS2 scans were acquired in the ion trap using fast scan rate on precursors with 2-7 charge states and quadrupole isolation mode (isolation window: 0.7 m/z) with higher-energy collisional dissociation (HCD, 30%) activation type. Dynamic exclusion was set to 30 s. The temperature of ion transfer tube was 300°C and the S-lens RF level was set to 30. Raw data processing and quantification Label-free quantification (LFQ) was performed using Proteome Discoverer 2.4 (Thermo Scientific) based on the composite database: P. papillatum’s predicted proteome, and mitochondrial ORFs, Raw files were searched with SequestHT using Trypsin as the enzyme, allowing up to three missed cleavages. Peptide length was set to 6–144 amino acids, with precursor ion mass tolerance at 20 ppm, fragment mass tolerance at 0.5 Da, and a minimum of one peptide identified. Carbamidomethyl (C) was a fixed modification, while Acetyl (N-terminus), Met-loss (N-terminus), and oxidation of Met were dynamic modifications. A target/decoy strategy and 1.0% FDR were calculated using Percolator. Data were imported into Proteome Discoverer 2.4, and features were detected using the Minora Feature Detector algorithm. The area-under-the-curve (AUC) for aligned ion chromatograms was calculated to determine relative abundances.ProteomicsJeremy G. WidemanPARTIALCenter for Mechanisms of Evolution, Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, Arizona, USADiplonemaDiplonema Papillatum10.1101/2025.07.16.665091yuping.poh@asu.eduCenter for Mechanisms of Evolution, Biodesign Institute, School of Life Sciences, Arizona State UniversityUnited StatesfalseSubcellular proteomics of model diplonemid, Paradiplonema papillatumThe genome of diplonemid Paradiplonema papillatum, like many other protist organisms, encodes a vast swathe of hypothetical proteins, for which no annotation is currently available, and confounds interpretations that can be drawn from molecular analysis. As a result, many outstanding questions remain for this clade of heterotrophic unicellular flagellates, including their evolutionary divergence and modes of nutrition. To rectify this, we initiated subcellular proteomics on P. papillatum to resolve localization for approximately 5,000 proteins of this model diplonemid. We ultimately predict 2,435 proteins to 22 cellular compartments of this organism, for which we performed additional validation via a series of endogenous tagging procedures across new predictions. For the first time, we resolve the cell membrane of P. papillatum, showing an accumulation of previously identified carbohydrate active enzymes. Our topological reconstructions for these enzymes suggest exposure to extracellular space and supports recent proposals for the breakdown of plant and algal cell wall carbohydrates in the world’s oceans as a means of nutrition. In turn we localize various metabolic enzymes of the cell, demonstrating broad amino acid processing capabilities within the mitochondrion, along with new glycosomal-localized carbohydrate enzymes, which strengthen claims for ancestral compartmentalisation for certain steps within the modified peroxisomes shared between sister-clades kinetoplastids and diplonemids. This dataset constitutes a proteomic resource from which to explore the unique cell biology of diplonemids and clarify developmental trends throughout their evolutionary differentiation.2025-10-232025-06-17PXD065121NEWT:3555NEWT:71647NEWT:330879NEWT:35554NCBITaxon:12939NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:295546NCBITaxon:2719036NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:309807NEWT:10245NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:351581NEWT:43186NEWT:428146NEWT:498257NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:661410NEWT:638632NEWT:263737NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:5702NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:383379NEWT:1401257NEWT:418106NEWT:10029NEWT:913645NEWT:44491NEWT:641809NEWT:27933NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7719NEWT:7959NEWT:2261NEWT:868565NEWT:31156NEWT:3112NEWT:79329NEWT:4442NEWT:192875NEWT:1214915NEWT:12637NEWT:59729NEWT:2164133NEWT:295105NEWT:108061NEWT:60711NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:160621NEWT:212790NEWT:931281NEWT:4432NEWT:5762NEWT:5763NEWT:658457NEWT:1310161NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NEWT:2246NEWT:2652724NEWT:1912919NEWT:5757NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:752555NEWT:498211NEWT:5759NEWT:347256NEWT:1736231NEWT:5518NEWT:398007NEWT:1527468NEWT:1392NEWT:498217NEWT:498216NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:1367269NEWT:145458NEWT:633149NEWT:1149786NEWT:10239NEWT:68895NEWT:44685NEWT:161934NEWT:4897NEWT:1148NEWT:5744NEWT:5508NEWT:3329NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1235816NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1287689NEWT:1390NEWT:11557NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:3711NEWT:935293NEWT:375146NEWT:270643NEWT:2065263NEWT:1772NEWT:118698NEWT:1616117NEWT:263NEWT:118696NEWT:52283NEWT:11988NEWT:284812NEWT:8175NEWT:400667NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:459758NEWT:3702NEWT:1245466NEWT:243277NEWT:1246791NEWT:7067NEWT:118694NEWT:586722NEWT:2850NEWT:118691NEWT:33548NEWT:274NEWT:7070NCBITaxon:287NEWT:408172NEWT:408170NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:1436733NEWT:460519NEWT:1247411NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:333760NEWT:56636NEWT:1513458NEWT:1233681NCBITaxon:40559NEWT:506599NEWT:2853422NEWT:81077NEWT:84588NEWT:1679718NEWT:480NEWT:65349NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:128017NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NEWT:34613NCBITaxon:4932NEWT:109760NEWT:943274NEWT:644223NEWT:235443NEWT:108458NEWT:5936NEWT:272623NEWT:272624NEWT:320637NEWT:886559NEWT:55521NEWT:983964NEWT:118499NEWT:11706NEWT:32644NEWT:527796NEWT:1130798NEWT:55529NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1116234NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:3983NEWT:203696NEWT:352472NEWT:1071661NEWT:1202532NEWT:360094NEWT:470NEWT:41364NEWT:5911NEWT:1313NEWT:1218097NCBITaxon:50557NEWT:39251NEWT:39491NEWT:212142NCBITaxon:5811NEWT:29491NEWT:101841NEWT:2014887NEWT:33952NEWT:153009NEWT:261756NEWT:571256NEWT:40483NEWT:63366NEWT:63367NEWT:688333NEWT:215402NCBITaxon:548681NEWT:318586NCBITaxon:5820NEWT:1547NEWT:272634NEWT:198107NEWT:9031NEWT:1872122NEWT:1308NEWT:7091NEWT:1034015NEWT:760568NEWT:141262NEWT:108931NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:97477NEWT:7111NEWT:347515NEWT:172269NEWT:9534NEWT:6269NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:8694NEWT:4093NEWT:4096NEWT:8692NEWT:2903NEWT:185579NEWT:941442NEWT:536088NEWT:13076NEWT:1226408NEWT:7108NEWT:40479NEWT:43989NEWT:4076NEWT:142615NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:552NEWT:4084NEWT:63577NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:634955NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:13095NEWT:162425NEWT:216257NEWT:102169NEWT:208893NEWT:9986NEWT:4054NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:8655NEWT:5147NEWT:98351NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:207559NEWT:407821NCBITaxon:2NEWT:85057NEWT:985076NEWT:568708NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:267671NEWT:28532NEWT:353152NEWT:8496NEWT:7165NEWT:1257118NEWT:27448NEWT:40674NEWT:1194669NEWT:51329NEWT:243230NEWT:443906NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:1328388NEWT:104395NEWT:9557NEWT:9315NEWT:6289NEWT:377586NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:9554NEWT:118562NEWT:725NEWT:7137NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:596153NEWT:229533NEWT:1836NEWT:214053NCBITaxon:10298NEWT:80863NEWT:90675NEWT:105231NEWT:104385NEWT:74649NEWT:52638NEWT:57075NEWT:206672NEWT:4097NEWT:8697NEWT:8695NEWT:884204NEWT:71139NEWT:9785NEWT:6279NEWT:1123869NE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