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Method Article
Ute Bertsche
IMIT - Microbial Genetics - University of Tuebingen
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Peptidoglycan (PGN) is ubiquitous in nearly all bacterial species. The PGN sacculus protects the cells against their own internal turgor making PGN one of the most important targets for antibacterial treatment. Within the last sixty years PGN composition has been intensively studied by various methods. The breakthrough was the application of HPLC technology on the analysis of muropeptides. However, preparation of pure PGN relied on a very time consuming method of about one week. We established a purification protocol for both Gram-positive and Gram-negative bacteria which can be completely performed in plastic reaction tubes yielding pure muropeptides within 24 hours. The muropeptides can be analyzed by UPLC-MS, allowing their immediate determination. This new rapid method provides the feasibility to screen PGN composition even in high throughput, making it a highly useful tool for basic research as well as for the pharmaceutical industry.
Keywords
peptidoglycan isolation, UPLC-MS, high throughput analysis, S. aureus
Figure 1
Figure 2
The authors declare no conflict of interest.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
*Table 2* *Muropeptides of _E. coli_ Nissle 1917 analyzed by UPLC-MS* This table summarizes the muropeptides of _E. coli_ Nissle 1917 that could be detected by UPLC-MS. The sum formula to each mass is given. Each muropeptide contains one to three stem-peptides, each consisting of L-Ala – D-Glu – mDAP – D-Ala (– Gly). The length of each of the stem peptides is stated. In some cases Penta stem-peptides with Gly on position 5 are present. Loss of GlcNAc or acetyl is shown as -GlcNAc and -Acetyl. The muropeptides of the main peaks are highlighted in bold. The original references for these peaks are given.
- supplement0.pdf
*Table 1* *Muropeptides of _S. aureus_ SA113 analyzed by UPLC-MS* This table gives a summary of all muropeptides of _S. aureus_ SA113 that could be detected by UPLC-MS. All structures were drawn by ChemDrawUltra (PerkinElmer) which automatically calculates the mass of the molecule and the sum formula. The latter is given. Each muropeptide contains one to six stem-peptides, each consisting of L-Ala – D-Glx – L-Lys – D-Ala (– D-Ala). In general, Glx is Gln. Non-amidated Glu is indicated separately. The length of each of the stem peptides is stated. The amount of Gly residues building the interpeptide bridges is given as the sum of all Gly residues present in each muropeptide. In rare cases one Gly is replaced by one Ala. Changes on the saccharide moiety (loss or addition of GlcNAc; O-Acetylation; rare non-reduced MurNAc) are shown. The muropeptides of the main peaks are highlighted in bold. The original references for these peaks are given. n.d. … not determined
- supplement0.pdf
*Table 4* *Midipreparation for UPLC/MS or HPLC/MS analysis* Protocol for midipreparation for several UPLC/MS runs, or HPLC/MS analysis, or additional peak collecting for further research.
- supplement0.pdf
*Table 7* *Troubleshooting*
- supplement0.pdf
*Table 6* *UPLC gradient*
- supplement0.pdf
*Table 3* *Solutions and buffers for peptidoglycan preparation and UPLC/MS analysis*
- supplement0.pdf
*Table 5* *Minipreparation in 96 well plate for UPLC/MS analysis* This protocol results in enough material for one analysis per sample on UPLC. Always cover the samples properly with a foil to avoid evaporation or mixing of samples!
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An open repository of community-contributed protocols sponsored by Nature Research.
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Method Article
Ute Bertsche
IMIT - Microbial Genetics - University of Tuebingen
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 16 Dec, 2014
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
Peptidoglycan (PGN) is ubiquitous in nearly all bacterial species. The PGN sacculus protects the cells against their own internal turgor making PGN one of the most important targets for antibacterial treatment. Within the last sixty years PGN composition has been intensively studied by various methods. The breakthrough was the application of HPLC technology on the analysis of muropeptides. However, preparation of pure PGN relied on a very time consuming method of about one week. We established a purification protocol for both Gram-positive and Gram-negative bacteria which can be completely performed in plastic reaction tubes yielding pure muropeptides within 24 hours. The muropeptides can be analyzed by UPLC-MS, allowing their immediate determination. This new rapid method provides the feasibility to screen PGN composition even in high throughput, making it a highly useful tool for basic research as well as for the pharmaceutical industry.
Figure 1
Figure 2
The authors declare no conflict of interest.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
*Table 2* *Muropeptides of _E. coli_ Nissle 1917 analyzed by UPLC-MS* This table summarizes the muropeptides of _E. coli_ Nissle 1917 that could be detected by UPLC-MS. The sum formula to each mass is given. Each muropeptide contains one to three stem-peptides, each consisting of L-Ala – D-Glu – mDAP – D-Ala (– Gly). The length of each of the stem peptides is stated. In some cases Penta stem-peptides with Gly on position 5 are present. Loss of GlcNAc or acetyl is shown as -GlcNAc and -Acetyl. The muropeptides of the main peaks are highlighted in bold. The original references for these peaks are given.
- supplement0.pdf
*Table 1* *Muropeptides of _S. aureus_ SA113 analyzed by UPLC-MS* This table gives a summary of all muropeptides of _S. aureus_ SA113 that could be detected by UPLC-MS. All structures were drawn by ChemDrawUltra (PerkinElmer) which automatically calculates the mass of the molecule and the sum formula. The latter is given. Each muropeptide contains one to six stem-peptides, each consisting of L-Ala – D-Glx – L-Lys – D-Ala (– D-Ala). In general, Glx is Gln. Non-amidated Glu is indicated separately. The length of each of the stem peptides is stated. The amount of Gly residues building the interpeptide bridges is given as the sum of all Gly residues present in each muropeptide. In rare cases one Gly is replaced by one Ala. Changes on the saccharide moiety (loss or addition of GlcNAc; O-Acetylation; rare non-reduced MurNAc) are shown. The muropeptides of the main peaks are highlighted in bold. The original references for these peaks are given. n.d. … not determined
- supplement0.pdf
*Table 4* *Midipreparation for UPLC/MS or HPLC/MS analysis* Protocol for midipreparation for several UPLC/MS runs, or HPLC/MS analysis, or additional peak collecting for further research.
- supplement0.pdf
*Table 7* *Troubleshooting*
- supplement0.pdf
*Table 6* *UPLC gradient*
- supplement0.pdf
*Table 3* *Solutions and buffers for peptidoglycan preparation and UPLC/MS analysis*
- supplement0.pdf
*Table 5* *Minipreparation in 96 well plate for UPLC/MS analysis* This protocol results in enough material for one analysis per sample on UPLC. Always cover the samples properly with a foil to avoid evaporation or mixing of samples!
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