Computational protocol: Protection against Experimental Cryptococcosis following Vaccination with Glucan Particles Containing Cryptococcus Alkaline Extracts

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Protocol publication

[…] Samples were processed for analysis by LC-MS/MS as previously described (). Briefly, for each sample, 50 µg protein was electrophoresed into a 10% Mini-Protean Tris-glycine extended (TGX)-PAGE gel (Bio-Rad Laboratories) in Tris-glycine–SDS buffer, stained with Coomassie, destained with water, and removed as a gel slice. Gel slices were cut into 1- by 1-mm pieces, reduced in a 45 mM solution of 1,4-dithiothreitol (DTT) at 50°C for 30 min, alkylated in 100 mM iodoacetamide for 30 min, and digested with 2 ng/µl of sequencing-grade trypsin (Sigma) in 0.01% ProteaseMAX surfactant (Promega)–50 mM ammonium bicarbonate at 37°C for 18 h. The supernatant of each sample was then placed in a 0.5-ml microcentrifuge tube. The gel slices were further extracted with 80:20 acetonitrile–1% formic acid, combined with the supernatants of each sample, and completely dried in a SpeedVac.Tryptic peptide digests were reconstituted in 25 µl 5% acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid and separated using a NanoAcquity ultraperfomance liquid chromatography system (Waters Corporation, Milford, MA). In brief, a 3.0-µl injection was loaded in 5% acetonitrile containing 0.1% formic acid at 4.0 µl/min for 4.0 min onto a 100-µm-inner-diameter (i.d.) fused-silica precolumn packed with 2 cm of 5-µm (200-Å) Magic C18AQ resin (Bruker-Michrom, Auburn, CA) and eluted using a gradient at 300 nl/min onto a 75-µm-i.d. analytical column packed with 25 cm of 3-µm (100-Å) Magic C18AQ particles to a gravity-pulled tip. The solvents were A (water–0.1% formic acid) and B (acetonitrile–0.1% formic acid). A linear gradient was developed from 5% solvent A to 35% solvent B in 60 min. Ions were introduced by positive electrospray ionization via liquid junction into a Q Exactive hybrid mass spectrometer (Thermo Scientific). Mass spectra were acquired over m/z 300 to 1750 at 70,000 resolution (m/z 200), and data-dependent acquisition selected the 10 most abundant precursor ions for tandem mass spectrometry by higher-energy collisional dissociation fragmentation using an isolation width of 1.6 Da, collision energy of 27, and a resolution of 17,500.Raw data files were peak processed with Proteome Discoverer (version 1.3; Thermo Scientific) or Mascot Distiller (version 2.4; Matrix Science, Inc., Boston, MA) prior to database searching with the Mascot server (version 2.4) against the C. neoformans var. grubii database (National Center for Biotechnology Information). Search parameters included both trypsin specificity with two missed cleavages and no enzymatic specificity. The variable modifications of oxidized methionine, pyroglutamic acid for N-terminal glutamine, deamidation of aspargine and glutamine, N-terminal acetylation of the protein, and a fixed modification for carbamidomethyl cysteine were considered. The mass tolerances were 10 ppm for the precursor and 0.05 Da for the fragments. Search results were then loaded into the Scaffold viewer (Proteome Software, Inc., Portland, OR) for peptide/protein validation. MS data were analyzed with Mascot software using two search criteria: a conventional search based on tryptic digestion sites and an expanded search in which no digestion enzyme was specified. Proteins were identified with Scaffold software (Proteome Software, Inc.), and probabilities were assigned by using the Protein Prophet algorithm; identifications were accepted if they could be established at >95% probability for at least two peptides. The Mascot Distiller average quantitation method was used, in which the precursor intensities of the three most abundant peptides are used to compute a protein’s relative abundance in a sample. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Distiller, Mascot Server
Application MS-based untargeted proteomics
Organisms Saccharomyces cerevisiae, Mus musculus, Cryptococcus gattii VGIII, Cryptococcus neoformans, Homo sapiens
Diseases Cryptococcosis, Infection, Lung Diseases