Trypsin Digestion and IGP Enrichment

Proteins (500 μg) were reduced using 10 mM DTT at 56 °C for 45 min and alkylated with 20 mM iodoacetamide in the dark for 30 min at room temperature. The solutions were diluted 8-fold in 40 mM ammonium bicarbonate, and sequencing grade trypsin (Beijing Shengxia, China) was added to the solution at 1:40 (w/w), followed by incubation overnight at 37 °C. The peptides were desalted using a C18 Sep-Pak column. Twenty micrograms of desalted peptides were reserved for peptide detection, and the remaining peptides were subjected to the enrichment of IGPs using the Oasis MAX extraction cartridges (Waters), as previously described [18 (link)].