Zorbax eclipse plus c18 rapid resolution high definition column

LC–MS analyses were performed using an Agilent TOF spectrometer (G6550A) with an electrospray ionization (ESI) interface. The converted products produced by enzymatic reactions were separated on a ZORBAX Eclipse Plus C18 Rapid Resolution High Definition column (Agilent, 10 cm × 2.1 mm, 1.8 μm particle size) using an Agilent 6200 series UHPLC system consisting of a binary pump, autosampler, multicolumn thermostat, and diode array detector. The column was eluted isocratically with a solvent of water/acetonitrile/formic acid (90:10:0.1, by volume) at a column temperature of 40 °C and a flow rate 0.3 mL min⁻¹. All samples used in analyses were dissolved in absolute ethanol after removing methanol and were injected in a total volume of 2 μL. LC flow was directly injected into a mass spectrometer equipped with a Dual AJS ESI source. Sample ionization was performed in negative mode using the following conditions: drying gas, nitrogen (14 L min⁻¹, 200 °C); nebulizer gas, nitrogen (35 psi); capillary voltage, 3.5 kV; capillary temperature, 350 °C. Spectra were recorded over an m/z range of 50–3000 with an accumulation rate of 2 spectra/s. Data were processed using Mass Hunter Workstation Acquisition software. The chemical structure was drawn and the molecular masses of its fragments were calculated using ChemDraw Professional v 15.1.

Peptidoglycan was extracted from exponential-phase cells (OD₆₀₀ of ∼0.8) and then digested with mutanolysin as described previously (50 (link)). The resulting soluble muropeptides were reduced with sodium borohydride and separated by reverse-phase UHPLC (RP-UHPLC) with a 1290 chromatography system (Agilent Technologies) and a Zorbax Eclipse Plus C₁₈ Rapid Resolution High Definition column (100 by 2.1 mm with a particle size of 1.8 μm; Agilent Technologies) at 50°C using ammonium phosphate buffer and a methanol linear gradient as described previously (51 (link)). Muropeptides were identified according to their retention times by comparison with an L. lactis muropeptide reference chromatogram (51 (link)). The different muropeptides were quantified by integration of the peak areas, and the percentage of each peak was calculated as the ratio of its area over the sum of all peak areas. The peptidoglycan cross-linking index was calculated according to methods described previously (52 (link)), as follows: (1/2 Σ dimers + 2/3 Σ trimers + 3/4 Σ tetramers)/Σ all muropeptides.