Molecular mass markers

Moringa oleifera seeds were collected from trees at the Campus do Pici of the Federal University of Ceará – UFC (Ceará, Brazil) under authorization (number: 47766) of the Chico Mendes Institute for Conservation of Biodiversity – ICMBio. A voucher specimen (N EAC34591) was deposited in the Prisco Bezerra Herbarium (UFC). The yeasts C. albicans (ATCC 10231), C. parapsilosis (ATCC 22019), C. krusei (ATCC 6258), and C. tropicalis (clinical isolate) were obtained from the culture collection of the Laboratory of Emergent and Reemergent Pathogens – LAPERE, Department of Pathology and Legal Medicine, UFC. Rabbit blood was obtained from animals of colonies maintained at the UFC. Human blood samples (ABO system) were obtained from healthy donors in the blood bank HEMOCE (Hemotherapy Center of Ceará). Experimental protocols were approved by the Ethics Committee of UFC, Brazil (protocol number: 77/2016).
Molecular mass markers, chromatographic matrices, IPG buffer, and immobilized pH gradient gel strips were obtained from GE Healthcare Life Sciences (New York, NY, United States). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, United States).

DM64 (20 µg) and myotoxin II were dissolved in 20 mM HEPES pH 7.5 and incubated at 25°C for 15 min (1:1 and 1:2 mol/mol). The noncovalent complex formed between both proteins was stabilized with BS³ (bis(sulfosuccinimidyl)suberate, Thermo Fisher Scientific) for 90 min at 25°C. The reaction was stopped by quenching in 20 mM ammonium bicarbonate. Protein (5–10 µM final concentration) to cross-linker ratios ranging from 1:500 to 1:2,800 mol/mol (the latter corresponding to 1:20 w/w) were tested. DM64 and myotoxin II controls were individually submitted to the same experimental procedure, using the higher protein to cross-linker ratio. Aliquots (10%) of each sample were analyzed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS-PAGE) under reducing conditions (12%T, silver staining) (Laemmli, 1970 (link); Heukeshoven and Dernick, 1985 (link)). Molecular mass markers were from GE Healthcare. The remaining aliquots (90%) were injected on a Superdex 200 HR 10/30 column (GE Healthcare) to remove excess BS³ and protein aggregates. Finally, the samples were digested in solution with Lys-C endopeptidase (1:100 E:S, w/w) and trypsin (1:50 E:S, w/w) as previously described (Bastos et al., 2020 (link)). The experiment was run in duplicate.