Cross-Linking of Plasma Adsorption Samples

For cross-linking of MAC to the surface of S. pyogenes, we used pooled normal human plasma adsorbed onto the surface of S. pyogenes bacteria (Happonen et al., 2019; Hauri et al., 2019). Briefly, the S. pyogenes M1 serotype strain SF370 from the American Type Culture Collection (ATCC; strain reference 700294) and its derived ΔM1 mutant lacking the major surface antigen, the M1 protein, was grown at 37°C 5% CO₂ to mid-exponential phase (OD_{620 nm} ∼0.4–0.5) in Todd–Hewitt (TH) broth supplemented with 0.3% (w/v) yeast extract. The cells were harvested by centrifugation (1,900 × g, 10 min, 22°C), washed once with phosphate-buffered saline [PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride (Sigma)], recentrifuged (1,900 × g, 5 min, 22°C), and resuspended to an approximate concentration of 1 × 10⁹ colony forming units ml^–1. For plasma adsorption, 400 μl of pooled normal human plasma from healthy donors (Innovative Research) was supplemented with a final concentration of 10 μM argatroban (Sigma) to prevent any plasma clotting and was mixed with 100 μl of bacteria with subsequent incubation at 37°C for 30 min at 500 rpm.

SF370 and ΔM1 bacteria with adsorbed plasma proteins–including the MAC–were harvested by centrifugation (1,900 × g, 5 min, 22°C), washed twice with PBS, and finally resuspended in 200 μl of PBS for cross-linking. For cross-linking, heavy/light disuccinimidyl-suberate cross-linker (DSS-H12/D12, Creative Molecules Inc.) resuspended in 100% dimethylformamide (Sigma) was added to final concentrations of 0, 0.25, 0.5, 1.0, and 2.0 mM in duplicates, and the samples were incubated at 37°C for 1 h at 900 rpm. The cross-linking reaction was quenched with a final concentration of 50 mM ammonium bicarbonate (Sigma) at 37°C for 15 min at 900 rpm. The MAC complex adsorbed to the bacterial surface was released by limited proteolysis with 2 μG trypsin (Promega) at 37°C for 1 h at 800 rpm prior to cell debris removal by centrifugation (1,900 × g, 15 min) and subsequent supernatant recovery. Two hundred microliters of the supernatant containing the MAC was recovered, and any remaining bacteria were killed by heat inactivation (85°C, 5 min) prior to sample preparation for MS.