Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063659/20250213KM.xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063659/20250212_KM1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063659/20250212_KM2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063659/20250212_KM4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063659/20250212_KM5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063659/20250212_KM6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD063659/20250212_KM3.rawprimaryOK200smoreno@uga.eduKatherine MoenMass SpectrometryBottom-up proteomicsDvsfLc-ms/msSercahttps://www.ebi.ac.uk/pride/archive/projects/PXD063659Parasites were collected as previously described for DVSF crosslinking including the enrichment of membrane proteins step. After freeze-thaw, initial resuspension of pellets, and removal of soluble proteins, the pellets enriched with membrane proteins were lysed for 5 minutes on ice in lysis buffer (50 mM HEPES pH 7.4, 50 mM NaCl, 1% NP-40, and mini complete protease inhibitor [CAT: 11836170001]) with periodic vortexing to facilitate lysis. Lysates were centrifuged at 21,000 x g for 5 minutes at 4C. After washing 30 µL of Pierce α-HA magnetic beads (Cat. No. 88836) three times with lysis buffer, clarified lysates from 4 × 10⁸ parasites were added to the beads and incubated overnight at 4 °C. The beads were collected, and unbound proteins were removed. The beads were then washed with the lysis buffer 3 times before boiling with 1 x Laemmli in lysis buffer for western blot testing or the beads were frozen dry and sent for LC-MS/MS analysis.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. SERCA-HA DVSF HA-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.2026-03-312025-05-06PXD063659NEWT:1773NEWT:3555NEWT:1182590NEWT:10090NEWT:749200NEWT:35554NEWT:4120NEWT:5693NEWT:347515NEWT:1216979NEWT:307972NEWT:92867NEWT:990346NEWT:544496NEWT:5334NEWT:145953NEWT:257309NEWT:284812NEWT:115104NEWT:43330NEWT:67825NEWT:44544NEWT:13076NEWT:544404NEWT:3702NEWT:8839NEWT:4232NEWT:1736309NEWT:4113NEWT:7227NEWT:11298NEWT:885318NEWT:4081NEWT:876138NEWT:554NEWT:5691NEWT:260710NEWT:106592NEWT:237561NEWT:9913NEWT:10036NEWT:4100NEWT:7574NEWT:1351NEWT:1076NEWT:6763NEWT:7215NEWT:380394NEWT:272563NEWT:1639NEWT:188229NCBITaxon:79857NEWT:746360NEWT:6239NEWT:135588NEWT:135622NEWT:6915NEWT:9986NEWT:101510NEWT:3880NEWT:58002NEWT:9103NEWT:4577NEWT:146479NEWT:1000589NEWT:145943NEWT:85962NEWT:160488NEWT:317447NEWT:3635NEWT:7955NCBITaxon:2NEWT:7959NEWT:2261NEWT:3197NEWT:9615NEWT:884019NEWT:4565NEWT:1264690NEWT:169963NCBITaxon:38727NEWT:36329NEWT:34305NEWT:59729NCBITaxon:183674NEWT:626528NEWT:139927NEWT:4558NEWT:9606NEWT:367830NEWT:243230NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NEWT:470NCBITaxon:79824NCBITaxon:4563NEWT:3218NEWT:5759NEWT:9838NCBITaxon:9615NEWT:1736231NEWT:1193501NEWT:6287NEWT:6326NEWT:9796NEWT:2762NEWT:5476NEWT:562NEWT:260707NEWT:287NEWT:10117NEWT:10116NEWT:1280NEWT:1836NEWT:29760NEWT:260705NEWT:1148NEWT:4932NEWT:70448NEWT:9825NEWT:3603NEWT:698936NEWT:39946NEWT:11676NEWT:9823NEWT:100226NCBITaxon:6073NEWT:4896NEWT:6279NEWT:7370NEWT:573NEWT:6282NEWT:70910009-0006-0831-4284