Ultimate 3000 lc system

Samples were loaded onto an Ultimate 3000 LC system (Dionex, Thermo Scientific) as described (Gonzalez-Lozano et al., 2020 (link); Hondius et al., 2021 (link); van der Spek et al., 2021 (link)). A generally used hippocampal synaptosomes library created in-house with MaxQuant Software was used to annotate proteins. Spectra were annotated against the Uniprot mouse reference database.
Data quality control and statistical analysis were performed by using the downstream analysis 1024 pipeline for quantitative proteomics (Koopmans, 2020 (link); MS-DAP version 0.2.6.3; for up to date version see https://github.com/ftwkoopmans/msdap). Outliers were removed when the variation among replicates was too large as observed by deviating distribution plots, or in the case of disturbed protein detection as observed by altered retention time plots. This led to a final sample size of n = 9 for the control (FC/CTL fear conditioning with saline) condition and n = 8 for CHX treated samples (FC/CHX fear conditioning with cycloheximide).

Fermentation was performed in 50 ml Erlenmeyer flasks in 10 ml media in triplicates. Fresh spore-suspension was added to 10 ml pre-culture medium (Yeast extract 3.6 g/l and peptone 10 g/l) to a final concentration of 2 × 10⁵ spores/ml. Pre-culture was incubated at 25 °C, 200 rpm and 2.5 cm amplitude for 48 h (KS 4000 I control, IKA). For the pH controlled fermentation, the mycelia pellets from the pre-culture were transferred to Erlenmeyer flasks with cotton stoppers and 10 ml production media [34 (link)] containing 100 g/l glucose, 2 g/l (NH₄)₂SO₄, 0.15 g/l KH₂PO₄, 0.15 g/l K₂HPO₄, 0.1 g/l MgSO₄∙7H₂O, 0.1 g/l CaCl₂∙2H₂O, 0.005 g/l NaCl, 0.1 g/l ZnSO₄, 0.005 g/l FeSO₄·7H₂O and 30 g/l CaCO₃(for pH maintenance). Flasks were incubated at 25 °C, 200 rpm. The supernatant obtained from each fermentation culture was prepared as described by [35 (link)]. 1 ml sample from each flask was taken at day 5. 50 μl 50% H₂SO₄ was added and the mixture was heated to 80 °C and incubated for 15 min. The samples were then cooled down to the room temperature followed by centrifugation at 8000 rpm for 1 min. The supernatant was filtered through 0.45 μM HPLC-grade regenerated cellulose membrane filters for HPLC analysis. 250 μl of the filtrate were analyzed for the content of sugars and organic acids by HPLC (Dionex Ultimate 3000-LC system) with the conditions described in [31 (link)].

The 10- and 15-kDa bands present on SDS-PAGE gel and Western blot were excised from gel and PVDF membrane, respectively. Proteins were extracted and trypsin digested, using the method described by Shevchenko et al. [45 (link)]. The digested proteins were analyzed by an Ultimate 3000 LC System (Dionex, USA) coupled to an HCTultra PTM Discovery System (Bruker Daltonics Ltd., U.K.) at the Proteomics Research Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology, Thailand. The Bruker Daltonics Data Analysis version 4.0 (Bruker Daltonics Ltd., U.K.) was used to analyze raw mass spectrometric data. The MASCOT software (Matrix Science, UK) was used to search the obtained MS and MS/MS data against ~15,000 genome-derived predicted proteins of P. insidiosum (unpublished data).

These procedures were performed as previously described [19 (link), 20 (link)]. Briefly, protein spots were excised, reduced, alkylated and trypsinized overnight at 37°C. After extraction and washing, nanoscale LC separation of tryptic peptides was performed using an Ultimate 3000 LC System (Dionex, USA) that was coupled to an ESI-Ion Trap MS (HCTultra PTM Discovery System, Bruker, Germany). The analyzed MS/MS data were submitted to a database search that used a local Mascot server with specific parameters. The Mascot results were used as queries for protein identification searches in the National Center for Biotechnology Information (NCBI) database. Proteins that met our criteria for “identified proteins” exhibited ≥1 peptide with an individual Mascot score of p < 0.05.

An HCTUltra PTM Discovery System (Bruker Daltonics Ltd., Bremen, Germany) coupled with an UltiMate 3000 LC System (Dionex Ltd., Camberley, UK) was used to analyze peptides in each sample. The peptide samples were separated on a nanocolumn (Acclaim PepMap 100 column 75 um × 5 cm) using reversed-phase high-performance liquid chromatography. Two eluents were used. Eluent A was 0.1% formic acid and eluent B was 80% ACN in water containing 0.1% formic acid. A 5–55% eluent B gradient was used to elute peptides at a constant flow rate of 0.30 μL/min for 30 min. Electrospray ionization was carried at 1.6 kV using the CaptiveSpray. Nitrogen, a drying gas, was applied with a flow rate of about 50 L/h. Collision-induced-dissociation product ion mass spectra were achieved using nitrogen gas as the collision gas. Mass spectra (MS) and MS/MS spectra were collected in positive-ion mode at 2 Hz over the (m/z) range 150–2200. The collision energy was adjusted to 10 eV as a function of the m/z value. The LC-MS analysis of each sample was performed in triplicate.

LC–MS/MS analysis was performed using an LTQ Orbitrap Velos hybrid-mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) and Xcalibur version 2.0.7 software provided with the UltiMate 3000 LC system (Dionex, LC Packings, Sunnyvale, CA, USA). Relative quantitation of unlabeled proteins was performed using Progenesis LC–MS data analysis software (version 4.1, Nonlinear Dynamics, Durham, NC, USA). To identify peptides, peak lists were created using a Progenesis LC–MS and we used MASCOT (v2.4.1, Matrix Science, London, UK) to search Rattus norvegicus protein sequences in the UniProt Knowledgebase (UniProtKB/Swiss-Prot) database (version May 2013; 7,853 entries). The search parameters were as follows: trypsin digestion with two missed cleavages permitted; variable modifications, protein N-terminal acetylation, oxidation of methionine, carbamidomethylation of cysteine, N-terminal carbamylation, and phosphorylation of serine, threonine, and tyrosine; peptide-mass tolerance for MS data, ±5 ppm and fragment mass tolerance, ±0.5 Da. The estimation of false discovery rate (FDR) was accomplished using a MASCOT decoy database. We used 1% FDR as the cutoff to export results from the analysis, and peptides that yielded a peptide ion score ≥25 were considered as identified.