Effects of incubation temperature and acetonitrile amount on microwave-assisted tryptic digestion of proteins.

The effects of incubation temperature and acetonitrile (ACN) amount on microwave-assisted tryptic digestion of horse skeletal muscle myoglobin (MYG) and bovine serum albumin (BSA) were investigated. Microwave-assisted tryptic digestion was performed on BSA or MYG solutions containing different amounts (0, 10, and 20%) of ACN for different times (10, 20, 30, 40, and 50 min) at different temperatures (25, 37, and 55 °C). Conventional overnight tryptic digestion was also conducted with gentle mixing at 37 °C for 16 h. Digested samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Similar sequence coverage (SC) values were obtained under most conditions except when the protein sample solutions were digested with 20% ACN at 55 °C, which provided the lowest SC for both MYG and BSA for all investigated digestion times. Considering the missed cleavage (MC) ratios for 50-min microwave-assisted digestion, the highest MC ratio, (i.e., lowest trypsin activity) was observed for the digestion condition of 20% ACN at 55 °C for both proteins, while the lowest MC ratio was observed with 0% ACN at 25 °C for MYG and with 0% ACN at 55 °C for BSA. Conventional overnight tryptic digestion at 37 °C provided more completely cleaved peptides than 50-min microwave-assisted tryptic digestion at the same temperature.