Sample Preparation for Mass Spectrometry

The sample preparation for mass spectrometry was done as described [4 (link),15 (link)]. Briefly, the samples were denatured in 8 M urea-100 mM ammonium bicarbonate (both Sigma), and the cysteine bonds reduced with 5 mM tris(2-carboxyethyl)phosphine (Sigma) (37 °C, 30 min). The cysteines were alkylated with 5 mM iodoacetamide (Sigma) (22 °C, 60 min), and the samples subsequently digested using sequencing-grade lysyl endopeptidase (37 °C, 2 h) (Wako). The samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M and followed by digestion using trypsin (Promega) (37 °C, 18 h). 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.