Muropeptide LC-MS Analysis and Data Processing Protocols for Core Facilities.

Peptidoglycans are major components of the bacterial cell envelope, providing a dynamic scaffold that is intimately involved in the growth, development, and survival of all prokaryotes. As peptidoglycan biosynthesis is the target of most commercially available antibiotics and peptidoglycan fragments (muropeptides) are a source of signaling molecules that modulate multiple processes, assessment of peptidoglycan structure and its associated muropeptides is an important component of many microbiology-based research endeavors. Inasmuch, many core facilities need to be able to measure these materials in routine fashion using LC-MS protocols. We provide our current methods of muropeptide sample processing and data analysis, using the muropeptides of Borrelia burgdorferi (spirochete) as an example. Muropeptides were prepared in standard fashion and reduced with either NaBH4 or NaBD4 in alkaline borate buffer. Samples were then either analyzed directly or after solid phase extraction employing a Shimadzu LCMS9030 interfaced with a Nexera LC-40 UHPLC. Chromatographic conditions were fully compatible with the requirements of a core facility with respect to columns (C18), mobile phases (acidified water and acetonitrile), and run times (10 min/run). The resulting datasets were then moved to open source MS-DIAL for initial interrogation and database generation, followed by Byonic for automated structural identification. Implementing a muropeptide analysis protocol is straightforward and can provide an additional source of samples for core proteomics and/or metabolomics facilities.