Microcons

The AAF sample was treated with lysis buffer (100 mM Tris–HCl, 1% SDS, 50 mM DTT), according to the standard MED-FASP protocol^{39 (link)}. The protein concentration was established by measuring absorbance at 280 nm (Multiskan Thermo) using a μDrop plate. 100 µg of proteins were digested on 10 kDa Microcons (Merck-Millipore) successively by Lys-C, trypsin, and chymotrypsin. The fractions collected after the centrifugation were subjected to the final clean up with StageTips according to the protocol described by Rappsilber and collaborators^{40 (link)}. For each desalting step, 10 µg of the peptides were taken and desalted on StageTip (3 layers of 3 M Empore C18 exchange discs in each tip). Fragmentation spectra were recorded on the Triple TOF 5600 + (Sciex) spectrometer (SCIEX, Framingham, MA) coupled with the EkspertMicroLC 200 Plus System (Eksigent, Redwood City, CA). Registration was performed in the data-dependent acquisition (DDA) mode, and the protein identification was conducted in the Peaks Studio program against the Annelida database (Uniprot).

C. albicans cells treated with different AAF concentrations (25, 50, 100 µg mL⁻¹) were lysed, and released proteins were prepared as described above. Before digestion, all samples were dissolved in a urea-containing solution (8 M urea in 0.1 M Tris–HCl pH 8.5). The protein concentration was established by measuring absorbance at 280 nm (Multiskan Thermo) using a μDrop plate. 100 µg of protein from each of the samples were digested separately with trypsin on 10 kDa Microcons (Merck-Millipore) according to the standard FASP protocol and prepared for mass spectrometry analysis^{43 (link)}. Final clean up preceding mass spectrometry measurements was done with StageTips according to the protocol described by Rappsilber and collaborators^{40 (link)}. For each desalting step, 10 µg of the peptide were taken and desalted on StageTip (3 layers of 3 M Empore C18 exchange discs in each tip).