Purification of SpAED/S18L, Edman degradation and MALDI-TOF mass spectrometry

Overnight bacterial cultures of S. aureus WY110 (pSpA_ED or its derivatives) were diluted 1:100 into 4 liters of TSB and grown to OD₆₀₀ 2. Staphylococci were sedimented by centrifugation at 8,000 × g for 10 min. Bacteria were suspended in 30 ml of Tris-buffer, 0.5 (vol/vol) 0.1 mm sterilized glass beads were added and peptidoglycan was broken with 15 × 1 min pulses in a bead-beating instrument (MP Biomedicals). Samples were centrifuged at 7,000 × g for 10 min to sediment glass beads. The supernatant was transferred to another tube and centrifuged at 33,000 × g for 1 hr at 4°C. The membrane sediment was suspended in 30 ml RIPA buffer and incubated for 1 hr with rotation. RIPA extract was centrifuged at 33,000 × g for 1 hr at 4°C. The supernatant was removed and subjected to affinity chromatography. Two ml 50% suspension of IgG sepharose (GE Healthcare) was loaded onto each column. The column bed was washed once with 7 ml 0.1 M glycine (pH 3.0), twice with 14 ml 50 mM Tris-HCl (pH 7. 5) and once with 10 ml RIPA buffer. RIPA membrane extracts were loaded onto the column followed by two washes with 14 ml RIPA buffer and once with 10 ml 50 mM Tris-HCl (pH 7. 5). Proteins were eluted by adding four times 1 ml 0.1 M glycine (pH 3.0) to the column and immediately neutralizing the eluate with 25 µl of 1.5 M Tris (pH 8.8). For Edman degradation, the purified SpA_ED precursors were 10-fold concentrated via Amicon Ultra-0.5 ml Centrifugal Filters (10 kD cut off). Proteins were separated on 15% SDS-PAGE, electro-transferred to PVDF and stained with Coomassie-Brilliant Blue. Bands of interest were excised and subjected to Edman degradation (Alphalyse, Inc, CA, USA). For MALDI-TOF mass spectrometry analysis, 1 µl of SpA_ED sample was mixed with 1 µl of 10 mg/ml sinapic acid, dried on the Bruker MTP 384 plate, and examined in a Bruker Autoflex Speed MALDI-TOF mass spectrometer in the linear positive-ion mode using peptide standards for calibration.