Venom Protein Purification and Digestion

Venom protein concentration was measured by bicinchoninic acid assay (Thermo Fisher Scientific) and an aliquot of 10 µg of protein per sample processed with SP3 beads (Thermo Fisher Scientific) following the protocol of Hughes et al. [43 (link)]. Briefly, venoms were centrifuged, and the cysteine disulfide bonds were reduced and alkylated with dithiothreitol (40 mM final) and iodoacetamide (120 mM final), respectively. SP3 beads were added to the samples to bind the proteins and the beads were washed three times with 70% ethanol. The beads were then incubated with 0.4 µg of trypsin/lysC mixture (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate overnight at 37 °C. The peptides formed by the proteolysis were captured onto the beads by adding pure acetonitrile, and the beads were washed three times with acetonitrile before eluting the peptides with 2% DMSO in water. The samples were acidified to pH 4 with formic acid. Samples were vacuum dried and suspended in 15 µL of loading buffer (1% acetonitrile, 0.05% trifluoroacetic acid in water).