Name: Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics (Video) | JoVE Journal
Uploaded: 2020-10-13T15:00:00.000+00:00
Duration: 9 min 9 s
Description: Discover semi-quantitative peptidoglycan analysis using liquid chromatography mass spectrometry and bioinformatics. This computational method enables detailed profiling of bacterial cell wall composition with enhanced accuracy.

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5 minutes

The authors would like to thank Dr. Jennifer Geddes-McAlister and Dr. Anthony Clarke for their contributions in refining this protocol. This work was supported by operating grants from CIHR awarded to C.M.K (PJT 156111) and a NSERC Alexander Graham Bell CGS D awarded to E.M.A. Figures were created on BioRender.com.

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<li>Quintela, J. C., Caparros, M., de Pedro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variability+of+peptidoglycan+structural+parameters+in+Gram-negative+bacteria%5BTitle%5D)+AND+%22FEMS+Microbiology+Letters%22%5BJournal%5D)">Variability of peptidoglycan structural parameters in Gram-negative bacteria.</a> FEMS Microbiology Letters. 125, 95-100 (1995).</li>
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<li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muropeptides+in+Pseudomonas+aeruginosa+and+their+role+as+elicitors+of+%CE%B2-lactam-antibiotic+resistance%5BTitle%5D)+AND+%22Angewandte+Chemie+-+International+Edition%22%5BJournal%5D)">Muropeptides in Pseudomonas aeruginosa and their role as elicitors of β-lactam-antibiotic resistance.</a> Angewandte Chemie - International Edition. 55, 6882-6886 (2016).</li>
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</ol>

The authors declare no conflicts of interest.

This protocol describes a method to purify peptidoglycan from bacterial cultures, process for LC-MS detection and analyze composition using bioinformatic techniques. Here, we focus on Gram-negative bacteria and some slight modification will be required to enable analysis of Gram-positive bacteria.
The preparation of muropeptides has remained virtually the same since it was first produced in the 1960s9,11,15...

Peptidoglycan (PG) is a defining characteristic of bacteria that serves to maintain cell morphology, while providing structural support for proteins and other cellular components1,2. The backbone of PG is composed of alternating &#946;-1,4-linked N-acetyl muramic acid (MurNAc) and N-acetyl glucosamine (GlcNAc)1,2. Each MurNAc possesses a short peptide bound at the &#7429;-lactyl residue that can be crosslinked to adjacent disaccharide-linked peptides (Figure 1A,B). This crosslinking produces a mesh-like structure that encompasses the entire cell and is often referred to as a sacculus (Figure 1C). During PG synthesis, precursors are generated in the cytoplasm, and transported across the cytoplasmic membrane by flippases. Precursors are subsequently incorporated into the mature PG by transglycosylase and transpeptidase enzymes, which produce the glycosidic and peptide bonds, respectively3. However, once assembled, there are numerous enzymes produced by the bacteria that modify and/or degrade the PG to carry out a number of cellular processes, including growth and division. In addition, various modifications of the PG have been shown to confer adaptations specific to the strain, growth conditions, and environmental stress, which have been implicated in cell signalling, antimicrobial resistance, and host immune evasion4. As examples, a common modification is the addition of a C6 acetyl group on the MurNAc that confers resistance by limiting access to the glycan &#946;-1,4 linkages to host-produced lysozyme enzymes which degrade PG4,5,6. In Enterococci, substitution of the terminal &#7429;-Ala of the peptide sidechain with &#7429;-Lac confers a greater resistance to the antimicrobial, vancomycin7,8.
The general procedure for PG isolation and purification has remained relatively unchanged since it was described in the 1960s9. Bacterial membranes are dissolved through heat treatment with SDS, followed by enzymatic removal of bound proteins, glycolipids, and remaining DNA. The purified intact sacculus can be subsequently digested into the individual components by hydrolysis of the &#946;-1,4 linkage between GlcNAc and MurNAc. This digestion produces GlcNAc-MurNAc disaccharides with any structural modifications and/or crosslinks intact and are called muropeptides (Figure 1B).
Compositional analysis of PG was initially conducted through high pressure liquid chromatographic separation (HPLC) to purify each muropeptide followed by manual identification of muropeptides10,11. This has since been superseded by liquid chromatography tandem mass spectrometry (LC-MS), which increases detection sensitivity and decreases the manual workload of purifying each individual muropeptide. However, the time consuming and complex nature of the manual identification of muropeptides has remained a limiting factor, reducing the number of studies conducted. In recent years with the emergence of &#8220;omic&#8221; technologies, automated LC-MS feature extraction has become a powerful tool, allowing for rapid detection and identification of individual compounds in complex samples from very large datasets. Once the features are identified, bioinformatic software statistically compares the variation between samples using differential analysis isolating even minimal differences among the complex dataset and displaying them graphically to the user. The application of feature extraction software for the analysis of PG composition has only just begun to be explored12,13,14 and coupled to bioinformatic analysis12. Unlike proteomic analysis which benefits from the readily available protein databases that predict peptide fragmentation allowing for fully automated identification, no fragmentation library currently exists for peptidoglycomics. However, feature extraction can be coupled with known and predicted structural databases to predict muropeptide identification12. Here we present a detailed protocol for the use of LC-MS-based feature extraction combined with a muropeptide library for automated identification and bioinformatic differential analysis of PG composition (Figure 2).

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C18 reverse phase column - AdvanceBio Peptide column (100 mm x 2.1 mm 2.7 &amp;micro;m)</td><td>Agilent</td><td></td><td>LC-MS data acquisition</td></tr><tr><td>Heating mantle controller, Optichem</td><td>Fisher</td><td>50-401-788</td><td>for 4% SDS boil</td></tr><tr><td>Heating Mantle, 1000mL Hemispherical</td><td>Fisher</td><td>CG1000008</td><td>for 4% SDS boil</td></tr><tr><td>Incubator, 37&amp;deg;C</td><td></td><td></td><td>for sacculi purification and MS sample prep</td></tr><tr><td>Leibig condenser, 300MM 24/40,</td><td>Fisher</td><td>CG121805</td><td>for 4% SDS boil</td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td></td><td>for lyophilization of sacculi</td></tr><tr><td>Magentic stirrer</td><td>Fisher</td><td>90-691-18</td><td>for 4% SDS boil</td></tr><tr><td>mass spectrometer Q-Tof model UHD 6530</td><td>Aglient</td><td></td><td>LC-MS data acquisition</td></tr><tr><td>microcentrifuge filters, Nanosep MF 0.2 &amp;micro;m</td><td>Fisher</td><td>50-197-9573</td><td>cleanup of sample before MS injection</td></tr><tr><td>Retort stand</td><td>Fisher</td><td>12-000-102</td><td>for 4% SDS boil</td></tr><tr><td>Retort clamp</td><td>Fisher</td><td>S02629</td><td>for 4% SDS boil</td></tr><tr><td>round bottom flask - 1 liter pyrex</td><td>Fisher</td><td>07-250-084</td><td>for 4% SDS boil</td></tr><tr><td>Sonicator model 120</td><td>Fisher</td><td>FB120</td><td>for sacculi purification</td></tr><tr><td>Sonicator - micro tip</td><td>Fisher</td><td>FB4422</td><td>for sacculi purification</td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td></td><td>sacculi wash steps</td></tr><tr><td>Ultracentrifuge bottles, Ti45</td><td>Fisher</td><td>NC9691797</td><td>sacculi wash steps</td></tr><tr><td>Water supply</td><td>City</td><td></td><td>for water cooled condenser</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chemdraw</td><td>Cambridgesoft</td><td></td><td>molecular editor for muropeptide fragmentation prediction</td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td>viewing lists of annotated muropeptides, abundance, isotopic patterns, etc.</td></tr><tr><td>MassHunter Acquisition</td><td>Aglient</td><td></td><td>running QTOF instrument</td></tr><tr><td>MassHunter Mass Profiler Professional</td><td>Aglient</td><td></td><td>bioinformatic differential analysis</td></tr><tr><td>MassHunter Personal Compound Database and Library Manager</td><td>Aglient</td><td></td><td>muropeptide m/z MS database</td></tr><tr><td>MassHunter Profinder</td><td>Aglient</td><td></td><td>recursive feature extraction</td></tr><tr><td>MassHunter Qualitative analysis</td><td>Aglient</td><td></td><td>viewing MS and MS/MS chromatograms</td></tr><tr><td>Prism</td><td>Graphpad</td><td></td><td>Graphing software</td></tr><tr><td>Perseus</td><td>Max Plank Institute of Biochemistry</td><td></td><td>1D annotation</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A998-4</td><td></td></tr><tr><td>Ammonium acetate</td><td>Fisher</td><td>A637</td><td></td></tr><tr><td>Amylase</td><td>Sigma-Aldrich</td><td>A6380</td><td></td></tr><tr><td>Boric acid</td><td>Fisher</td><td>BP168-1</td><td></td></tr><tr><td>DNase</td><td>Fisher</td><td>EN0521</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>27001-500ML-R</td><td></td></tr><tr><td>LC-MS tuning mix - HP0321</td><td>Agilent</td><td>G1969-85000</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Mutanolysin from Streptomyces globisporus ATCC 21553</td><td>Sigma-Aldrich</td><td>M9901</td><td></td></tr><tr><td>Nitrogen gas (&gt;99% purity)</td><td>Praxair</td><td>NI 5.0UH-T</td><td></td></tr><tr><td>Phosphoric acid</td><td>Fisher</td><td>A242</td><td></td></tr><tr><td>Pronase E from Streptomyces griseus</td><td>Sigma-Aldrich</td><td>P5147</td><td></td></tr><tr><td>RNase</td><td>Fisher</td><td>EN0531</td><td></td></tr><tr><td>Sodium azide</td><td>Fisher</td><td>S0489</td><td></td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>452890</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Fisher</td><td>BP166</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher</td><td>S318</td><td></td></tr><tr><td>Sodium Phosphate (dibasic)</td><td>Fisher</td><td>S373</td><td></td></tr><tr><td>Sodium Phosphate (monobasic)</td><td>Fisher</td><td>S369</td><td></td></tr><tr><td>Stains-all</td><td>Sigma-Aldrich</td><td>E9379</td><td></td></tr></tbody></table>

semi-quantitative analysis of peptidoglycan by liquid chromatography mass spectrometry and bioinformatics

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&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

Increased detection sensitivity of MS machinery coupled with high-powered peak recognition software has improved the ability to isolate, monitor, and analyze substance compositions of complex samples in very minute detail. Using these technological advancements, recent studies on peptidoglycan composition have begun to use automated LC-MS feature extraction techniques12,13,14,24 over older HPLC...

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

semi-quantitative-analysis-peptidoglycan-liquid-chromatography-mass

Research

JoVE Journal

Biochemistry

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Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

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Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

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Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

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Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

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Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Discover semi-quantitative peptidoglycan analysis using liquid chromatography mass spectrometry and bioinformatics. This computational method enables detailed profiling of bacterial cell wall composition with enhanced accuracy.

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

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