Phlebia brevispora BAFC633 proteome
; Chelaliche, Anibal Sebastian ; Rodríguez, María Daniela ; Zapata, Pedro Dario ; Fonseca, Maria Isabel Otros responsables
Fecha
Publicador:
Consejo Nacional de Investigaciones Científicas y Técnicas
Fecha de depósito:
18/04/2024
Fecha de creación:
27/02/2024
Clasificación temática
Resumen
This is the proteome (NS01 and NS02) and secretome (NS03 and NS04) of Phlebia brevispora BAF633 obtained from the mycellium of the fungus harvested in a liquid malt extract medium supplemented with L-tyrosine. The data was obtained via nanoHPCL MS/MS and by using the proteome discover software. This dataset contains the accesion number of every protein identified.
Métodos
For the whole proteome analysis, the 8 days mycelium culture of P. brevispora was first subjected to a cellular breakdown using liquid nitrogen. After this, protein extraction was performed: 500 mL of extraction buffer was added to the lysate, incubated for 1h at room temperature and then centrifuged to obtain the protein extract. For the secretome analysis, the supernatant was clarified to remove remaining cells using 0.22 mm filters (Chromafil®xtra). The protein concentration of each sample was determined using the Pierce™ BCA protein Assay Kit (Thermo Scientific). Each sample was then reduced with 10 mM dithiothreitol (DTT) at 56°C for 45 minutes and alkylated with a solution of 20 mM iodoacetamide at room temperature for 45 minutes in darkness. Finally, samples were precipitated with trichloroacetic acid for 2 h at 20°C and washed three times with cold acetone (4 °C). The obtained pellets were analyzed by a nanoHPCL EASY-nLC 1000 at the Center for Chemical and Biological Studies by Mass Spectrometry, University of Buenos Aires, Argentina (http://cequibiem.qb.fcen.uba.ar). The obtained pellets of both procedures were suspended in ammonium bicarbonate buffer (50 mM, pH 8) and digested with trypsin overnight. Samples then were freeze dried using SpeedVac and resuspended in 30ul of trifluoroacetic acid 0.1%. A final desalting process was carried out using a C18 Zip Tip (Merck). The samples were passed through an EASY-Spray Accucore (P/N ES801) column (C18, 2um, 100A, 75 um x 150 mm) using a temperature of 35°C, a flow of 300 nL/min and a gradient of water with 0.1% of formic acid (solution “A”) and acetonitrile with 0.1% of formic acid (solution “B”). Peptides were separated by using a gradient of 30% of B solution for 60 min and a gradient of 95% of B solution for 15 minutes. The mass spectra were obtained using a Q-exactive spectrometer (Thermo Scientific). The 12 most intense signal obtained in the Full-scan MS were selected for MS/MS spectra. All of these spectra were analyzed by Proteome Discoverer using Phlebia, Agaricus, Trametes and Pleurotus data bases, with 2 miscleavages allowed, a precursor ion mass tolerance of 10 ppm, a fragmented ion mass tolerance of 0.05Da and using Oxidation as a dynamic modification and Carbamidomethylation as a static modification.
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