Comprehensive Serum Proteome and Glycoproteome Analysis

Protein extraction and digestion was performed as previously described [30 (link)]. In brief, the serum protein was denatured in 8 M urea/ 50 mM NH₄HCO₃, then reduced with DTT, alkylated with IAM, digested with lysyl endopeptidase (Promega; Madison, WI, USA) for 4 h at 37 °C, and then incubated with trypsin (Promega) overnight at 37 °C with shaking. The digested peptides were acidified with 10% trifluoroacetic acid to pH < 3, collected by centrifugation and purified using Oasis HLB cartridges (Waters; Milford, MA, USA). Desalted peptides were subjected to LC–MS/MS analysis for proteomics. N-glycopeptides were enriched by MAX column (Waters) using desalted peptides, and subjected to LC–MS/MS analysis for glycoproteomics as described previously [35 (link)].