Carboxypeptidase y

Pandonodin thermostability was tested with and without reducing reagents. Forty microliter samples of aqueous pandonodin at 5.8 mg/mL was prepared with the peptide alone, with 20 mM dithiothreitol (DTT), or with 5% β-mercaptoethanol (BME). These were then each split into 4 × 10 μL samples, where one sample was not further processed, one sample was digested with carboxypeptidase, one sample was heat-treated, and the last sample was heat-treated and then digested with carboxypeptidase. All samples after addition of reducing reagent were allowed to incubate at room temperature for 1 hour before any further processing. For samples that required no further processing steps, the samples were placed at 4 °C until all samples were ready for analysis by LC-MS. Heat-treatment was conducted at 95 °C for 2 hours in a thermocycler with a heated lid. Carboxypeptidase digestion was carried out in 100 μL reactions with 50 mM sodium acetate buffer, pH 6, 1 unit of carboxypeptidase B (Sigma-Aldrich) and 1 unit of carboxypeptidase Y (Affymetrix) for 3 hours at room temperature. All other samples were diluted to the same concentration with sodium acetate buffer before analysis. Two microliters of each sample was analyzed by LC-MS, where the first two minutes were diverted to waste.

Dried thrombin-cleaved lasso peptides and linear peptides were digested with 0.5 U carboxypeptidase B (Sigma-Aldrich) and 0.5 U carboxypeptidase Y (Affymetrix) in carboxypeptidase digestion buffer (50 mM sodium acetate, pH 6.0) for 3 h at room temperature. Digested samples were further analyzed by MALDI-MS as described above.