Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230317KM.xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_9.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063619/20230715_KM_10.rawprimaryOK200smoreno@uga.eduKatherine MoenMass SpectrometryBottom-up proteomicsSterptavadi-ipLc-ms/mshttps://www.ebi.ac.uk/pride/archive/projects/PXD063619Subcellular fractionation was carried out as previously described [71, 109]. Following a 1 hr incubation with 50 μM biotin at 37°C, parasites were collected, needle lysed out of host cells, and filtered through an 8 µm nuclepore membrane [71]. Parasites were then washed with BAG, counted, and lysed by grinding with silicone carbide on ice for 30-sec intervals with 30-sec pauses in between for a total grinding time of approximately 2 min, in order to keep organelles intact. Parasites and silicon carbide were resuspended in lysis buffer (50 mM KCl, 4 mM MgCl2, 0.5 mM EDTA, 20 mM HEPES-KOH pH 7.2, 125 mM sucrose, mini complete protease inhibitor, 12 µg/mL DNase, 12 µg/mL RNase, and 8 µg/mL nocodazole). Silicon carbide was removed through a series of low-g centrifugations. Fractions were collected after each centrifugation step (Fig S6F). Pellets and supernatants were collected, and supernatants re-spun for the following centrifugation until pellet (P3) fraction, which was homogenized and mixed with the 20% layer of an Optiprep [CAT: M1248-250] gradient for a total volume of 12 mL (Fig S6F). The gradient was top loaded and then centrifuged at 50,000 x g for 1 hour using a SW-41Ti rotor in an Optima XE-100 Ultracentrifuge. 24-500 μL fractions were collected from the top of the gradient and labeled as (starting from the top) 1a, 1b, 2a, 2b, etc. until 12b (the bottom fraction). Fractions of interest were diluted fivefold, re-centrifuged at 100,000xg for 1 hr, and resuspended in lysis buffer and protease inhibitor to remove potentially contaminating iodixanol from the Optiprep. Fractions of interest from both TgPDIA3-TID-3HA-GEEL and FBXO14-TID were lysed in RIPA buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% NP-40 in 50 mM HEPES pH 7.5) as previously described [110] and incubated with Streptavidin magnetic beads for 1hr at RT. Proteins were eluted from the beads using a buffer with excess biotin (20 mM Biotin 1% SDS and 25 mM Tris, pH 7.4) at 75°C for 30 min. Co-IP eluates were sent to the University of Nebraska Proteomics & Metabolomics Facility for LC-MS/MS analysis. 2 biological replicates each for S4 and the combined ER gradient fractions 6a, 6b, 7a, and 7b were sent. Results were analyzed using Scaffold to determine those proteins enriched in the ER fractions in TgPDIA3-TurboID cell line compared to S4 in FBXO14 cytosolic controls. Samples were submitted to the Proteomics and Metabolomics Facility, Nebraska Center for Biotechnology, University of Nebraska-Lincoln and analyze for mass spectrometry (MS) as previously described [112]. Briefly, an aliquot of 37.5 µL of sample was added to 12.5 µL of 4X reducing NuPAGE LDS gel (Thermo Fisher Scientific, Waltham, MA) sample buffer at 5 mM dithiothreitol (DTT) and incubated at 95°C for 10 min. The samples were loaded and run on a Bolt 12% Bis-Tris-Plus gel (Thermo Fisher Scientific) in MES SDS running buffer to clean them and concentrate the proteins into the top of the gel. The gel was then fixed in methanol:acetic acid:water (40:10:50), and stained with Colloidal Coomassie blue G-250. The gel containing proteins was excised and destained in 50% acetonitrile (ACN), 50 mM ammonium bicarbonate. The proteins were reduced in 100 mM ammonium bicarbonate with DTT at 10 mM. The reducing buffer was removed, and proteins were alkylated with iodoacetamide at 10 mM. Proteins were digested with 250 ng of trypsin overnight at 37°C. Peptides were extracted from the gel pieces, dried down, and re-dissolved in 5% acetonitrile, 0.2% formic acid. Each digest was run by nanoLC-MS/MS using a 2 h gradient on a Waters CSH 0.075 mm x 250 mm C18 column (Waters Corp, Milford, MA) feeding into a Thermo Orbitrap Eclipse mass spectrometer.PrideAll MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.7). Mascot was set up to search the cRAP_20150130.fasta (125 entries); and ToxoDB-59_TgondiiGT1_AnnotatedProteins_20221003 (8,460 sequences) assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.060 Da and a parent ion tolerance of 15.0 PPM. Carbamidomethyl of cysteine was set as a fixed modification. Deamidated of asparagine and glutamine, oxidation of methionine was specified in Mascot as variable modifications. Scaffold (version Scaffold_5.2.2; Proteome Software Inc., Portland, OR) was used to validate LC-MS/MS-based peptide and protein identifications. Hits were identified with 99% protein threshold, 95% peptide threshold, and a minimum peptide number of 2. Statistical analysis was conducted using Fisher’s exact test with Benjamini-Hochberg multiple test corrections to identify significantly enriched proteins.ProteomicsSilvia MorenoCenter for Tropical and Emerging Global Diseases and Department of Cellular Biology, Moreno lab, University of Georgia, United States of AmericaPARTIALToxoplasma Gondii Gt1katherine.moen26@uga.eduNot availableUniversity of GeorgiaUnited StatesfalseA Protein Disulfide Isomerase Coordinates Redox Homeostasis and ER Calcium Regulation for Optimal Lytic Cycle Progression in Toxoplasma gondii. streptavadin-IP LC-MS/MSThe endoplasmic reticulum (ER) maintains an oxidative environment that promotes disulfide bond formation, a critical process for proper protein folding. Protein disulfide isomerases (PDIs) are ER resident enzymes that facilitate the formation, breakage, and rearrangement of disulfide bonds between cysteine residues, thereby stabilizing protein structures. Although PDIs are functionally diverse, they all contain at least 1 thioredoxin-like domain and mediate disulfide exchange through their conserved CXXC motifs. The Apicomplexan parasite, Toxoplasma gondii, infects approximately one third of the world population, posing a significant risk to immunosuppressed individuals and unborn fetuses. Its fast-replicating tachyzoite form engages in a lytic cycle, causing host tissue damage and contributing to pathogenesis. While approximately 26 PDIs are predicted to be present in T. gondii, their specific roles remain largely unexplored. In this study, we investigate TgPDIA3, a T. gondii PDI localized to the ER, along with several of its interacting protein substrates. We explore its role in ER redox activity and calcium sequestration and assess how these functions contribute to the parasite’s lytic cycle.2026-03-312025-05-05PXD063619NEWT:1773NEWT:3555NEWT:38783NEWT:8727NEWT:1182590NEWT:8726NEWT:2NEWT:157546NEWT:10090NEWT:935293NEWT:749200NEWT:35554NEWT:4120NEWT:5693NEWT:9417NEWT:347515NEWT:8724NEWT:1216979NEWT:307972NEWT:92867NEWT:8723NEWT:990346NEWT:544496NEWT:5334NEWT:145953NEWT:257309NEWT:5180NEWT:284812NEWT:115104NCBITaxon:1313NEWT:1081927NEWT:43330NEWT:67825NEWT:44544NEWT:13076NEWT:373995NEWT:544404NEWT:3702NEWT:8839NEWT:4232NEWT:990119NEWT:1736309NEWT:4113NEWT:7227NEWT:11298NEWT:7469NEWT:885318NEWT:171101NEWT:4081NEWT:876138NEWT:554NEWT:5691NEWT:98334NEWT:408170NEWT:260710NEWT:3708NEWT:106592NEWT:237561NEWT:9913NEWT:10036NEWT:4100NEWT:7574NEWT:1351NEWT:1076NEWT:6763NEWT:7215NEWT:803NEWT:8030NEWT:380394NEWT:272563NEWT:507601NEWT:1639NEWT:188229NEWT:4909NCBITaxon:79857NEWT:95648NEWT:746360NEWT:6239NEWT:1589NEWT:135588NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:95486NEWT:3880NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:1911079NEWT:1000589NEWT:145943NEWT:1902NEWT:85962NEWT:160488NEWT:317447NEWT:3635NEWT:7955NCBITaxon:2NEWT:7959NEWT:2261NEWT:3197NEWT:9615NEWT:884019NEWT:4565NEWT:1264690NEWT:169963NCBITaxon:38727NEWT:36329NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:9606NEWT:367830NEWT:157295NEWT:243230NEWT:931281NEWT:373153NEWT:7029NEWT:1283300NEWT:334747NEWT:470NCBITaxon:79824NCBITaxon:4563NEWT:3218NEWT:5759NEWT:9838NCBITaxon:9615NEWT:1736231NEWT:1193501NEWT:6287NEWT:2242NEWT:6326NEWT:9796NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:260707NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:29760NEWT:260705NEWT:80863NEWT:1148NEWT:4932NEWT:70448NEWT:9825NEWT:3603NEWT:698936NEWT:2759NEWT:39946NEWT:11676NEWT:9823NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:7370NEWT:573NEWT:6282NEWT:7091NEWT:11345060009-0006-0831-4284