High-throughput Automated Muropeptide Analysis (HAMA) Reveals Peptidoglycan Composition of Gut Microbial Cell Walls

Ya Chen Hsu, Pin Rui Su, Lin Jie Huang, Kum Yi Cheng, Chun Hsien Chen, Cheng Chih Hsu*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Peptidoglycan (PGN), a net-like polymer constituted by muropeptides, provides protection for microorganisms and has been a major target for antibiotics for decades. Researchers have explored host-microbiome interactions through PGN recognition systems and discovered key muropeptides modulating host responses. However, most common characterization techniques for muropeptides are labor-intensive and require manual analysis of mass spectra due to the complex cross-linked PGN structures. Each species has unique moiety modifications and inter-/intra-bridges, which further complicates the structural analysis of PGN. Here, we developed a high-throughput automated muropeptide analysis (HAMA) platform leveraging tandem mass spectrometry and in silico muropeptide MS/MS fragmentation matching to comprehensively identify muropeptide structures, quantify their abundance, and infer PGN cross-linking types. We demonstrated the effectiveness of HAMA platform using well-characterized PGNs from E. coli and S. aureus and further applied it to common gut bacteria including Bifidobacterium, Bacteroides, Lactobacillus, Enterococcus, and Akkermansia muciiniphila. Specifically, we found that the stiffness and strength of the cell envelopes may correspond to the lengths and compositions of interpeptide bridges within Bifidobacterium species. In summary, the HAMA framework exhibits an automated, intuitive, and accurate analysis of PGN compositions, which may serve as a potential tool to investigate the post-synthetic modifications of saccharides, the variation in interpeptide bridges, and the types of cross-linking within bacterial PGNs.

Original languageEnglish
Publication statusPublished - Jun 2023

Bibliographical note

Funding Information:
This research was supported by Ministry of Sciencee cahnndo Tlogy (MT), R.O.C. (GrOS ants MOST 108-2636-M-002-008-, 109-2636-M-002-005-, and 110-2.6-C36. H-M. -002-014-). Y acknowledged the financial support of “The Program of Research Performance Enhancement via Students Entering Ph.D. Programs Straight from an Undergraduate/Master’s Progarm” from Natioaniawl aTn Univer. sTihtye instrument support from the NTU Mass Spectrometry Platform was acknowledged.

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© 2023, eLife Sciences Publications Ltd. All rights reserved.


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