LC-MS analysis of peptidoglycan crosslinking activity.

This procedure was adapted from prior reports.^16,18 E. faecalis Lipid II (20 µM), PGT (0.5 µM), and bPBP (1 µM) were incubated in a 30 µL 1x reaction buffer (50 mM HEPES pH 7.5, 10 mM MgCl₂, 30% DMSO) at 30 ºC for 1 h unless otherwise indicated. The reactions were heat-quenched at 95 ºC for 3 min and allowed to cool to room temperature. Polymerized material was digested by incubating the mixture at 37 ºC for 3 h after the addition of 65 µL ddH₂O and 5 µL mutanolysin (from Streptomyces globisporus, 4000 U/mL). To reduce the muropeptide products, 50 µL sodium borohydride (10 mg/mL) was added and the mixture was incubated at room temperature for 30 min. The pH was adjusted to ~4 with 20% phosphoric acid and the samples were lyophilized to dryness overnight. The samples were resuspended in 20 µL ddH₂O and analyzed by LC-MS on an Agilent 6520 Q-TOF mass spectrometer with ESI-MS operating in positive mode. Muropeptide products were separated on a Waters Symmetry Shield RP18 column (5 µm, 3.9 × 150 mm) with the following method: flow rate = 0.5 mL/min, 100% solvent A (H₂O, 0.1% formic acid) for 5 min followed by a linear gradient of solvent B (acetonitrile, 0.1% formic acid) from 0 to 40% over 25 min. Molecular ions for the target muropeptide fragments were extracted from the total ion chromatogram.