2.3. Protein Extraction and Proteomics Analysis

Proteins were extracted from 300 µL overnight cultures (18–20 h of growth in YPD medium at 37 °C). These conditions were chosen for proteomic analysis as they gave a superior reproducibility in test pilot studies. Under these conditions, most of the cells remained in yeast form. Cell extracts were obtained by using glass beads in a Fast prep breaker, as described previously [47]. The supernatant was collected, and the protein concentration was measured using a Bradford assay. Protein extracts were precipitated with MeOH/chloroform and resuspended in 8 M Urea. Protein extracts (100 µg) were reduced with 10 mM DTT (Sigma-Aldrich) for 1 h at 37 °C followed by alkylation with 55 mM iodoacetamide (Sigma-Aldrich) for 1 h in the dark at room temperature. Proteins were digested with 1/50 (w/w) of recombinant trypsin (Roche Molecular Biochemicals, Mannheim, Germany) in 25 mM ammonium bicarbonate adjusted to pH = 8.5 and incubated overnight at 37 °C. Digested peptides were desalted and concentrated, as described before [48]. Peptides were analyzed by reverse phase liquid chromatography electrospray ionization tandem mass spectrometry (RP-LC-ESI-MS/MS) on a nano Easy-nLC 1000 (ThermoScientific, San Jose, CA, USA,) coupled to a Q-Exactive HF mass spectrometer (Thermo Scientific). Desalted peptides were concentrated by loading them on an Acclaim PepMap 100 column (Thermo Scientific, 20 mm × 75 μm inner diameter, 3 µm diameter C18 and 100 Å pore size). Peptides were separated and eluted on a C18 Picofrit column (Thermo Scientific Easy Spray Column, PepMap RSLC C18 500 mm × 75 μM inner diameter, 2 μM diameter, 100 Å pore size) with an integrated spray tip at a flow rate of 250 nL/min for 240 min. Buffer A (2% acetonitrile and 0.1% formic acid) and buffer B (0.1% formic acid on acetonitrile, gradient from 2 to 40%) were used. Peptides were detected with a Q-Exactive mass spectrometer at a m/z range of 350–2000 Da with a mass resolution of 60,000 and acquired using data-dependent acquisition (DDA). The 15 most abundant precursors with charges of 2–6+ (threshold 8 × 10³) were selected for higher energy collisional dissociation (HCD) fragmentation with a dynamic exclusion of 27 s. The normalized collision energy was 27%.

Peptide identification was carried out by using the Mascot v. 2.6.1 search engine (MatrixScience) through the Protein Discoverer 2.2 Software (Thermo Scientific) and the CGD21 database from http://www.candidagenome.org accessed on date 10 April 2018. The following parameters were used: tolerances of 10 ppm for precursor ions and 0.02 Da for MS/MS fragment ions, up to two missed cleavage sites from trypsin digestion and allowing optional methionine oxidation and fixed carbamidomethylation of cysteine. The acceptance criteria for protein identification were an FDR < 1% and at least one unique peptide identified with high confidence (percolator q-value < 0.01). Protein quantification was carried out, as previously described [48]. Protein extracts were treated for mass spectrometry at the Proteomics Unit of the Universidad Complutense de Madrid.

CGD was the main database used for the functional classification. GO Slim Mapper tool was used for the analysis by cellular component, while the ontological enrichment analysis by biological process was carried out by PathoYeastract, Rank by GO.