Purification and Identification of Staphylococcal PSMs

Fifty-microliters of overnight cultures of S. epidermidis or S. haemolyticus strains were inoculated into 5 ml of tryptic soy broth and aerobically cultured at 37°C for 18 h or 14 h. The culture supernatants were dried using a centrifugal evaporator. The precipitates were dissolved in 40% acetonitrile and the soluble fraction was dried. The precipitates were dissolved in water and analyzed in reversed phase HPLC using SOURCE 5RPC ST 4.6/150 column (GE Healthcare, Tokyo, Japan) and 50% acetonitrile in 0.1% trifluoroacetic acid for 3 min and a water/acetonitrile gradient in 0.1% trifluoroacetic acid from 50% to 90% acetonitrile for 20 min at a flow rate of 1 ml/min (600E, Waters, Milford, MA). Absorbance at 215 nm was measured using a 2998 Photodiode Array Detector (Waters). Each PSM species was identified by liquid chromatography/electrospray-ionization mass spectroscopy (LC 1100 series, Agilent Technologies, Santa Clara, CA; ESI-MS, Bio-TOFQ, Bruker Daltonics, Billerica, MA) and the predicted molecular masses of PSMs [25] (link), [27] . S. epidermidis PSMα and PSMδ were eluted at the same retention time in this assay condition.