Centrifugal concentrator

A mixture of 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate was used for de-staining the blue color from gel slides [13 ]. Venom proteins were reduced by 4mM DTT and incubated at 60°C for 15 min. The reduced proteins were further alkylated by 250 mM iodoacetamine (IAA) (Sigma-Aldrich, USA) and incubated at room temperature for 30 min in dark. The gel pieces were dehydrated by removing all solution and adding 100% ACN (Thermo Scientific, USA). For tryptic digestion, trypsin (Sigma-Aldrich, USA, T6567) in 50 mM ammonium bicarbonate (Sigma-Aldrich, USA) was added to rehydrate the gels, which were then incubated overnight at 37 ˚C. Peptide extraction was performed by adding 100% ACN and incubating for 15 min. The resulting solution was transferred into a new microcentrifuge tube and dried using a centrifugal concentrator (TOMY, Japan). The peptide mixtures were stored at -20°C prior to mass spectrometric analysis.

Ten micrograms of SABH was mixed with 2× protein loading buffer, containing 2× Laemmli sample buffer (Bio-Rad Laboratories, USA) and 2-mercaptoethanol, and denatured by heating at 65 °C for 10 min. The sample was subjected to one-dimensional SDS-PAGE, using 5% stacking and 12% resolving gels, and a mini-vertical electrophoresis system (Bio-Rad Laboratories, USA). The gels were stained with Coomassie brilliant blue G250. An individual lane of the SDS-PAGE gel was segmentally cut along its length into small pieces. To perform in-gel digestion, gel pieces were destained until colourless, using acetonitrile (50%) in 50 mM NH₄HCO₃. After the destaining solution had been removed, 10 mM dithiothreitol was added to the gel pieces, followed by incubation at 60 °C for 15 min. Proteins were alkylated using 55 mM iodoacetamide in 50 mM NH₄HCO₃ at room temperature for 30 min in the dark. Following the removal of all solution, the gel pieces were dehydrated by 100% ACN (Sigma-Aldrich, USA) and dried at room temperature. Protein digestion was performed overnight at 37 °C, using 0.1 mg/mL trypsin (Sigma-Aldrich, USA) in 50 mM ammonium bicarbonate. Peptides were extracted in 50% ACN. The resulting supernatant was transferred to microcentrifuge tubes and dried in a centrifugal concentrator (TOMY, Japan) at 45 °C.

MMAE (10 µM) and ADCs (equivalent to 10 µM MMAE) were prepared. To Control- and IF-ADC solution, 1.5 µM PLM was added and incubated for 2 h at 37 °C to see how much MMAE could be released from each ADC. Protein residue was removed by acetone precipitation. The supernatant was dried and solidified using a centrifugal concentrator (Tomy, Japan). Free MMAE from ADCs was measured using the Nexera X2 HPLC system (Shimadzu, Japan). Disodium phosphate (10 mM, pH 6.8) was used for mobile phase A, and acetonitrile was used for mobile phase B. Samples were separated using the CAPCELL PAK C18AQ column (3 μm polar, 4.6 mm i.d. × 100 mm, Shiseido) heated to 40 °C and equilibrated with 10% mobile phase A. MMAE was eluted using 52% mobile phase A and detected using detection wavelength 224 nm. Ibuprofen (Sigma) was used for an internal control, was eluted by 27% mobile phase A and was detected at the same detection wavelength as MMAE.