Aeris widepore column

Venom samples were redissolved in LCMS water and diluted to a concentration of 0.18 mg/mL. We determined venom protein concentration by the A280 reading from a NanoDrop Photospectrometer (ThermoFisher Scientific, Waltham, MA, USA). We then loaded 15 μg of venom protein onto a Prominence HPLC apparatus (Shimadzu Corp., Columbia, MD, USA). Samples were run through a Phenomenex Aeris widepore column (3.6 micron, C18, part # 00G-4482-AN) maintained at 25 °C. Samples were eluted with solution A as 0.1 mM TFA in water and solution B as 0.06 mM TFA in acetonitrile, and absorbance at 220 nm was measured over 150 min with the following run parameters: 5 min at 10% B, 110 min increasing from 10 to 55% B, 5 min increasing from 55 to 75% B, 5 min at 75% B, 5 min decreasing from 75–10% B, and 5 min at 10% B. Venom peak areas were quantified using Shimadzu Lab Solution v2 software, combining default peak calls by the iPeakFinder algorithm with manual curation of the peak baseline and filtering of peaks making up less than 0.1% total area as noise. Peaks were called uniquely per individual snake, by aligning the serial HPLC profiles from all samples from a single individual and calling peaks that were present. The relative abundance of HPLC peaks in each sample will be henceforth referred to as venom composition.