4.9. Peptidoglycan HPLC-MS Analysis

HPLC-MS experiments were focused on the separation and identification of individual products of enzymatic digestion of the peptidoglycan. Chromatographic analyses were carried out on an Agilent 1100 series instrument (Agilent Technologies, Santa Clara CA, USA) coupled with a diode-array detector (Agilent Technologies, Santa Clara CA, USA) and an ion-trap mass spectrometer amaZon SL equipped with an ESI ion source (Bruker Daltonics, Fremont, CA, USA). The chromatographic separation was performed on Poroshell 120 SB-AQ column (150 × 2.1 mm; 2.7 μm) (Agilent Technologies, Santa Clara CA, USA) at 35 °C. The mobile phase consisted of eluent A: 0.1% formic acid in water and eluent B: 0.1% formic acid in methanol at a flow rate of 0.2 mL min⁻¹. The gradient started at 1% of B, increased at a constant rate until 70% of B in 20 min, then held constant until 25 min, and then back to the initial 1%, and held constant until 40 min. The injected volume was 2.5 µL. The MS conditions were as follows: spray voltage, −5.0 kV; the pressure of nebulizer gas, 25 psi; the flow of dry gas, 10 L/min; the temperature of dry gas, 300 °C. The measurements were performed in Ultrascan mode in the range of m/z 70–2200. Structural analyses were based on MS data obtained in negative ion mode.