Lysyl endopeptidase c

Samples were denatured with 8 M Urea and reduced with 5 mM dithiothreitol (DTT) at 37 °C for 45 min, followed by alkylation with 40 mM chloroacetamide (CAA) for 45 min in the dark. The urea was then diluted to 1 M with 50 mM Triethylammonium bicarbonate buffer (TEAB), pH 8.0. Protein digestion was finished by using Lysyl endopeptidase C (Wako) with an enzyme-to-protein ratio 1:100 (w/w) for 3 h at 37 °C and continued with trypsin (Serva) at an enzyme-to-protein ration 1:50 (w/w) for overnight at 37 °C. Enzymically-digested samples were cleaned with C8 Sep-Pak (Waters), dried and stored at –20 °C for further analysis.

Cross-linked vacuoles were digested in solution. Proteins were denatured by incubation in 8 M urea in 50 mM tetraethylammonium bromide (TEAB), reduced with 5 mM dithiothreitol (DTT) for 60 min at 37 °C and alkylated with 40 mM chloroacetamide at room temperature for 30 min in the dark. Proteins were digested with Lysyl endopeptidase C (Wako) at an enzyme-to-protein ratio of 1:75 (w/w) at 37 °C for 4 h. After diluting with 50 mM TEAB to a final concentration of 2 M urea, the digestion was continued with trypsin (Serva) at an enzyme-to-protein ratio of 1:100 (w/w) at 37 °C overnight. Peptides were desalted with Sep-Pak C18 cartridges (Waters) and dried in a SpeedVac.