Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph—mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.