Staphylococcus aureus protein binding in human serum

Overnight cultures of S. aureus were diluted to OD600 = 1 and 1 ml of culture was resuspended in 500 μl of either PBS, human plasma, or human serum, and incubated rotating at 37 °C for 30 m. Suspensions were washed 3x with PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide. Cells were incubated with immobilized trypsin (ThermoFisher 20230), suspended in PBS supplemented with 500 40% sucrose and 20 mM Sodium Azide, at 37 °C for 2 h. cells were pelleted, and the supernatant containing protein fragments was analyzed by mass spectrometry to determine proteins.