Sequencing grade lysyl endopeptidase

Samples from cross-linking of purified proteins and cross-linking of plasma adsorption were denatured in 8 M urea–100 mM ammonium bicarbonate, and the cysteine bonds reduced with 5 mM tris(2-carboxyethyl)phosphine (37 °C, 30 min) and alkylated with 5 mM iodoacetamide (22 °C, 60 min). Samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, and sequencing-grade lysyl endopeptidase (37 °C, 2 h) (Wako Chemicals) followed by trypsin (37 °C, 18 h) (Promega) was added for protein digestion. Digested samples were acidified with 10% formic acid to a pH of 3.0, and the peptides were subsequently purified with C18 reverse-phase spin columns according to the manufacturer’s instructions (Macrospin columns, Harvard Apparatus). Dried peptides were reconstituted in 2% acetonitrile and 0.2% formic acid prior to MS analyses.

The cross-linked antibody-spike samples were denatured with 8 M urea - 100 mM ammonium bicarbonate, and the cysteine bonds reduced with a final concentration of 5 mM TCEP (37°C for 2h, 800 rpm) and subsequently alkylated with a final concentration of 10 mM iodoacetamide (22°C for 30 min, in the dark). The proteins were first digested with 1 µg of sequencing grade lysyl endopeptidase (Wako Chemicals) at 37°C, 800 rpm, 2h, diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, after which 1 µg sequencing grade trypsin (Promega) was added for further protein digestion (37°C, 800 rpm, 18 h). The samples were acidified to a final pH of 3.0 with 10% formic acid, and the peptides purified with C18 reverse phase spin columns according to the manufacturer’s instructions (Macrospin columns, Harvard Apparatus). The peptides were dried in a speedvac and reconstituted in 2% acetonitrile, 0.2% formic acid prior to mass spectrometric analyses.