Hypersil gold aq column

The peptidoglycan isolated from all four strains (wild-type, ΔltgA, ΔltgA^ltgA and ΔltgA^ltgAΔ30) was incubated for 16 hr in the presence of 10 µg of mutanolysin in 12.5 mM sodium phosphate buffer (pH 5.6) at 37°C (total reaction volume 150 µl). The reaction was stopped by boiling the samples for 3 min, and the supernatant containing the soluble muropeptides was collected after centrifugation at 16,000 ×g for 10 min. The supernatant was analyzed by reversed-phase HPLC using a Hypersil GOLD aQ column (5 μm particle size, 150 × 4.6 mm, flow rate 0.5 ml at 52°C, Thermo Fisher Scientific) with a mobile phase of H₂O-0.05% trifluoroacetic acid and a 25% acetonitrile gradient over 130 min. Muropeptides of interest were collected and identified by mass spectrometry as previously described (Williams et al., 2017 (link); Williams et al., 2018 (link)).

Chromatographic separation was achieved on a Thermo Scientific Hypersil Gold aQ column (1.9 μm, 50 × 2.1 mm) held at 40 °C. The column was eluted at 0.5 mL/min using mobiles phases containing 0.1% formic acid (v/v) in (A) Milli-Q water and (B) MeCN. The autosampler chamber was maintained at 10 °C and the injection volume was 1 μL. The reaction products (in both the oxidized and reduced states) were also analyzed using the BEH phenyl column and NH₄OH mobile phases as described in Section 4.5. The initial solvent composition was 5% B with a linear gradient to 30% B from 0.5 to 3 min, ramped to 95% B by 3.5 min, held at 95% B until 4 min, and followed by a linear gradient back to 5% B at 4.5 min. The column was then re-equilibrated with 5% B until 5 min.
Scanning experiments were performed in both +ESI and −ESI modes with preliminary scan ranges of m/z 48–1000 and m/z 800–1800, followed by a narrow scan range of m/z 900−1100. CID fragmentation experiments were performed on the dominant precursor ions in −ESI with CEs ranging from 40 to 100 eV and a scan range of m/z 48–1000.

Chemical composition was determined using Ultra High Performance Liquid Chromatography (UHPLC) modified as previously described [31 (link),43 ]. UHPLC analysis was performed using a Thermo Scientific UHPLC UltiMate 3000 (Waltham, MA, USA) with a Hypersil GOLD™ a Q column (100 × 2.1 mm i.d., 1.9 µm, Thermo Scientific™) and results were analyzed with Thermo Scientific™ LCQUAN™ software. Crude extracts of Al and Gg and standard solutions (oxyresveratrol, resveratrol, gallic acid and glabridin) were dissolved in methanol at concentrations of 1 and 10 mg/mL. Samples were filtered (0.20 mm, Millipore) and 1 μL was directly injected. Solvents for HPLC analysis were formic acid (0.1% v/v) in water as solvent A and formic acid (0.1% v/v) in methanol as solvent B, at a flow rate of 0.5 mL/min. These experiments used the following gradient: 30% B linear (0–4 min), 30–50% B linear (4–5 min), 50–70% B linear (5–8 min), 70–100% B linear (8–12 min), 100% B (12–15 min), and 30% B linear (15–18 min). An equilibrium period of 5 min was performed prior to the injection of subsequent samples. Chromatograms were recorded at 280 nm (glabridin and gallic acid), 305 nm (resveratrol), and 326 nm (oxyresveratrol), using the photodiode detector. Quantitative determination of compounds was performed using peak area with an external standard.