4.6. Peptidoglycan Purification and Analysis

PG samples were analyzed as described previously with some modifications [66,67]. Briefly, cells were pelleted and resuspended in 400 µL H₂O with 5% SDS and boiled for 1 h. Sacculi were washed twice with MilliQ water by ultracentrifugation (110,000 rpm, 30 min, 20 °C) and finally resuspended in 40 μL H₂O. The samples were treated with muramidase (100 μg/mL) for 16 h at 37 °C. Muramidase digestion was stopped by boiling for 15 min and coagulated proteins were removed by centrifugation (15 min, 14,000 rpm). For sample reduction, the pH of the supernatant was adjusted to pH 8.5–9.0 with sodium borate buffer, and sodium borohydride was added to a final concentration of 10 mg/mL. After incubating for 30 min at room temperature, pH was finally adjusted to 3.5 with orthophosphoric acid.

Detection and characterization of muropeptides by LC–MS was performed on an UPLC ™ system interfaced with a Xevo G2/XS Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA) equipped with an ACQUITY UPLC BEH C18 Column (130Å, 1.7 μm, 2.1 mm × 150 mm (Waters, USA)). Muropeptides were separated at 45 °C using a linear gradient from buffer A (formic acid 0.1% in water) to buffer B (formic acid 0.1% in acetonitrile) in an 18-min run, with a 0.25 mL/min flow. The QTOF–MS instrument was operated in positive ionization mode. Detection of muropeptides was performed by MS^E to allow for the acquisition of precursor and product ion data simultaneously, using the following parameters: capillary voltage at 3.0 kV, source temperature to 120 °C, desolvation temperature to 350 °C, sample cone voltage to 40 V, cone gas flow 100 L h⁻¹, and desolvation gas flow 500 L h⁻¹. Mass spectra were acquired at a speed of 0.25 s/scan. The scan was in a range of m/z 100–2000. Data acquisition and processing was performed using UNIFI software package (Waters Corp.). An in-house compound library built in UNIFI was used for detection and identification of muropeptides. Subsequent identification and confirmation of each muropeptide was performed by comparison of the retention-times and mass spectrometric data to known samples. Quantification was performed by integrating peak areas from extracted ion chromatograms (EICs) of the corresponding m/z value of each muropeptide and normalized to their molar ratio.

Main PG features were calculated as follows: percentage of monomers, dimers, trimers, and tetramers was calculated by adding the relative molar abundances of the different oligomers; overall crosslink was calculated as Dimers + (Trimers × 2) + (Tetramers × 3); percentage of anhydro muropeptides was calculated by adding the relative molar abundances of the different anhydro species; and average glycan chain length was calculated by dividing 100 by the percentage of anhydro muropeptides.