Centrivap centrifugal concentrator

A total of 5 mg crude venom powder was reconstituted in 0.1% TFA and centrifuged at 15,000× g at 4 °C for 10 min; the supernatant was then subjected to a Kromasil 300 RP-C18 column (250 × 4.6 mm, 5 μm; AkzoNobel) using a Waters E600 HPLC system (Waters, Milford, MA, USA). The venom proteins were separated at 1 mL/min using a linear gradient of the mobile phase A (0.1% TFA) and B (100% ACN) according to Gao et al. [62 (link)]: isocratically 10% B for 10 min, 10–15% B over 10 min, 15–45% B over 80 min, and 45–60% B over 50 min. The separation process was monitored at 215 nm. The eluted fractions were collected manually and concentrated in a CentriVap^® Centrifugal Concentrator (Labconco, Kansas, MO, USA).

Each sample was analyzed via the shotgun proteomics approach. In the first step, the samples were digested by trypsin. Disulfide bonds were reduced and alkylated by incubation of samples with 5 mM DTT (Sigma Aldrich, USA) for 1 h at 37°C with subsequent incubation in 15 mM iodoacetamide (Sigma Aldrich, USA) for 30 min in the dark at room temperature. For tryptic digestion, the samples were diluted with seven volumes of 50 mM ammonium bicarbonate and incubated for 16 h at 37°C with 400 ng of Trypsin Gold (1:50 ratio; Promega, USA). Tryptic peptides were desalted by solid-phase extraction using stage tips. Stage-tips were prepared according to Matamoros et al.: polypropylene Vertex pipette tips (200 μl; SSIbio, USA) were filled with four layers of C18 reversed-phase excised from Empore 3M C18 extraction disks (17 (link)). The desalted peptides were evaporated in a Labconco Centrivap Centrifugal Concentrator (Labconco, USA) and stored at –20°C prior to analysis.

Fresh venom was extracted from each snake using a 100 μL plastic pipette, then centrifuged to remove impurities for 15 min at 10,000× g 4 °C, lyophilized, equally pooled and stored at −80 °C until use. Three milligrams of venom powder was re-dissolved in 0.1% TFA and centrifuged for 15 min at 10,000× g, 4 °C, and the supernatant was automatically loaded onto a Kromasil C18 column (250 × 4.6 mm, 5 μm particle size, 300 Å pore size; AkzoNobel, Bohus, Sweden) and separated at a flow rate of 1 mL/min using a Waters E600 HPLC system (Waters, Milford, MA, USA). The whole process was performed with a linear gradient of mobile phase A (0.1% TFA in water) and B (100% ACN): 0–15% B for 30 min, followed by 15–45% B for 120 min and 45–70% B for 20 min. Protein detection was monitored at 215 nm. The fractions were collected manually and concentrated in a Labconco CentriVap^® Centrifugal Concentrator (Labconco, Kansas, MO, USA). Protein concentration was determined according to Bradford [55 (link)]. The proteins of each fraction were separated by 18% SDS-PAGE under reduced conditions, and the gels were stained in 0.2% Coomassie Brilliant Blue R-250 and imaged using a Tanon Imaging system (Tanon Science & Technology, Shanghai, China).