Hornodermoporus martius secretome
Here we present the secreted proteins by the fungus Hornodermoporus martius in a liquid medium, focusing on the proteolytic enzymes for fibrin degradation.
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Culture supernatant of H. martius LBM 224 was obtained on day 21 of culture in a liquid medium optimized for the production of fibrinolytic enzymes (composed of the following (in g/L): glucose, 35; meat peptone, 5; NaCl, 2; KH2PO4, 0.5; and MgSO4·7H2O, 0.5), it was filtered through a 0.22 um membrane. A reducing solution of Dithiothreitol was then added to the extract to a final concentration of 10mM per sample, incubating then for 45 min at 56 ± 1 ºC. Following the reduction, a solution of Iodoacetamide was added, reaching a final concentration of 20 mM and was incubated for 45 min at room temperature in darkness. For the precipitation, a quantity of trichloroacetic acid equal to one fifth of the protein extract of each sample was added, letting the proteins precipitate for 2 hs at -20 ºC. All samples were then centrifugate (10000 rpm, 10 min, 4 ºC) and the pellet was resuspended and washed 3 times with 500 μl of cold acetone (4 ºC). The obtained pellet of both analysis were suspended in an ammonium bicarbonate buffer (50mM, pH 8) along with 200 ng of trypsin (Promega®) to a final volume of 50 μl, incubating for 12 h. Lastly, the samples were lyophilized using SpeedVac and resuspended in 10 μl of formic acid 0,1% (v/v). A final desalting process was carried out using a C18 Zip Tip. All the obtained peptides were separated by a nanoHPCL EASY-nLC 1000 (Thermo Scientific) coupled with an electro spray EASY-SPRAY (Thermo Scientific). The samples were loaded in a C18 pre-column Acclaim PepMap (ID: 75 um, Length: 20 mm, Particle Size: 3 um) and then passed through a column EASY-SPRAY Accucore (ID: 75 um, Length: 150 mm, Particle size: 3 um) using a temperature of 35 ºC, a flow of 300 nl/min and a gradient of “A” solution (Water with 0,1% of formic ácid) and “B” Solution (Acetonitrile with 0,1% of formic acid). The peptides were separated by using a gradient of 0% to 35% of B solution for 110 min and a gradient of 35% to 95% of B solution for 1 min. The Full-Scan mass spectra were obtained using a Q-exactive spectrometer (Thermo Scientific). The 12 most intense ions obtained in the Full-scan MS were selected for dissociation into the high collision dissociation cell, getting as a result the MS/MS spectra of the selected ions. All of these spectra were analysed by the Proteome Discoverer Software (Thermo Scientific) using a Pleurotus data base with 2 miscleavages allowed, a precursor ion mass tolerance of 10 ppm, a fragmented ion mass tolerance of 0,05 Da and using Oxidation as a dynamic modification and Carbamidomethylation as a static modification.