Millex 0.22 μm

The wild-type FHbp ID 28 and site-specific mutant proteins were expressed from the T7 promotor using the E. coli plasmid pET21b (Novagen, Madison, WI) as described previously [5 (link), 27 (link)]. The recombinant proteins were purified by immobilized metal ion chromatography using Ni-NTA agarose (Qiagen, Valencia, CA) as described previously. Purified FHbps were dialyzed against PBS, sterilized by filtration (Millex 0.22 μm; Millipore, Billerica, MA), and stored at 4 °C prior to use. The protein concentrations were determined by UV absorbance (Nanodrop 1000, Wilmington, DE) based on the extinction coefficient calculated from the amino acid sequence [28] .

A UFLC Prominence Shimadzu LC device coupled to a DAD and MicrOTOF-Q III mass spectrometer (Bruker Daltonics, Billerica, MA, USA) was used for the analyses. A Kinetex C18 column (2.6 μm, 150 mm × 2.1 mm, Phenomenex) was applied, with a 1 μl injection volume, 0.3 ml/min flow rate, and 50 °C oven temperature. For the mobile phase, water (A) and acetonitrile (B) were used, to which 0.1% formic acid (v/v) was added. The gradient elution profile was the following: 3% B for 0–2 min, 3–25% B for 2–25 min, 25–80% B for 25–40 min, and 80% B for 40–43 min, followed by subsequent reconditioning conditions (5 min). The analyses were performed in negative and positive ion modes. Nitrogen was applied as the nebulizer gas at 4 bar and drying gas at 9 L/min. The capillary voltage was 3.5 kV. All of the extracts were prepared at 1 mg/ml, filtered (Millex0.22 μm, PTFE, Millipore), and injected in the chromatographic system.
The compounds were identified based on ultraviolet spectra, accurate mass, and fragmentation profile and compared with data in the literature. The molecular formulas were determined by accurate mass, considering errors up to 8 ppm and mSigma < 25.