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

1. Peptidoglycan sample preparation

1. Growth of bacterial cultures
   NOTE: The growth of the bacterial cultures will vary depending on the bacterial species and the growth conditions being examined. The experimental parameters to be tested will define the growth conditions.
   1. Grow bacterial cultures under growth conditions required for the bacterial strain and experimental design. Grow bacteria as triplicate cultures (biological replicates) i.e., three separate colonies per strain or growth condition.
      NOTE: Growth conditions and growth phase are known to have significant effects on PG composition^1,2,10. Great care must be taken to maintain consistency between cultures and replicates to ensure compositional changes are due to the experimental parameters and not experimental error.
   2. Rapidly cool the culture to 4 °C, collect by centrifugation (11,000 x g, 10 min, 4 °C) and freeze the cell pellet at -20 °C. Wash the cell pellet with pre-cooled 4 °C, 20 mM sodium phosphate pH 6.5 prior to freezing. Production of the frozen cell pellet should be done as quickly and as consistently between samples as possible to limit the activity of enzymes which could modify and/or degrade the PG during the collection process. Samples can be processed directly through the extraction process (section 1.2) without freezing; however, ensure that all samples are processed in a similar manner.
      NOTE: To ensure sufficient product for later steps, a significant sized wet cell pellet is used. This produces a large enough sacculi pellet, which is easily visualized and maintained during the repetitive washing steps (section 1.2.5 and 1.2.14) without significant loss of product. Depending on bacterium and growth conditions, this yield will likely vary. For the Gram-negative bacterium Pseudomonas aeruginosa, PAO1, 4 L of a 0.5 OD₆₀₀ culture produced a 3–4 g cell pellet and was sufficient to produce ~10 mg of purified sacculi (section 1.2.17)¹². This is a large excess of sacculi than is required for the LC-MS (section 2); however, it will aid in measurement accuracy (section 1.2.17) and normalization (section 1.3).
2. Extraction of peptidoglycan sacculi
   NOTE: The protocol for the extraction of PG is adapted from ref.^9,11,15. This protocol will extract the PG from individual bacterial cells as a whole sacculi, free of other cellular components. The protocol can be used with either Gram-negative or Gram-positive bacteria. However, for Gram-positive cells, adjustments may be necessary to isolate the thicker PG structure and to remove cell-wall associated polymers; such as, teichoic acids.
   1. Resuspend frozen cell pellets at approximately 1:10 of the original culture volume of 20 mM sodium phosphate pH 6.5. Perform this step at 4 °C (can be 1–8 mg wet cell pellet weight per mL of buffer^11,12).
      NOTE: To maintain the acetylation state of the PG, a pH of 6.5 or lower is required^15,16.
   2. Add cell suspension dropwise to boiling 8% sodium dodecyl sulfate (SDS) 20 mM sodium phosphate pH 6.5 for a final 1:1 volume (i.e., final concentration is 4% SDS), gently stirring in a round bottom flask outfitted with a water-cooled condenser. (Figure 2, step 2)
   3. Maintain a gentle boil for 30 min to 3 h with stirring to ensure complete membrane dissociation. Ensure that the resulting mixture is completely clear with no remaining cell clumps or viscosity. Longer boiling is preferred to guarantee complete dissociation.
   4. Allow it to cool to room temperature. The sample can be left at room temperature overnight.
      NOTE: When SDS is present, maintain samples at room temperature to keep SDS in the solution.
   5. Collect sacculi as a pellet through ultracentrifugation at 70,000 x g for 40 min (or the time required to completely sediment sacculi) at 25 °C.
   6. Repetitively wash sacculi by successive ultracentrifugation (section 1.2.5) and suspension in ~50 mL of room temperature 20 mM sodium phosphate pH 6.5 until the wash buffer has a SDS concentration ~0.001%. Typically, 5 to 7 washes are sufficient.
      NOTE: To test the concentration of the remaining SDS in the wash buffer, use the colorimetric dye, Stains-all¹⁷.
   7. Resuspend sacculi in 5–10 mL of room temperature 20 mM sodium phosphate pH 6.5.
   8. Sonicate the sample briefly (~40%, 50 W, 20 kHz, 20 s) at room temperature to disperse clumps.
      NOTE: Extended sonication will mechanically cause shearing of the PG structure¹⁸.
   9. Supplement the sample with 50 μg/mL each amylase, DNase, and RNase, 10 mM magnesium sulfate, and digest at 37 °C for 1 h with agitation or nutation.
      NOTE: Amylase digestion removes any remaining glycogen trapped within the sacculi¹¹.
   10. Add 100 μg/mL pronase and digest at 37 °C overnight with agitation or nutation and ~0.02% sodium azide.
       NOTE: Pronase digestion removes the enzymes added (from section 1.2.9) and removes lipoproteins that are covalently linked to the PG.
       CAUTION: Sodium azide is highly toxic and requires proper use/disposal methods.
   11. Ultracentrifuge at 70,000 x g for 40 min (or the time required to completely sediment sacculi) at 25 °C to remove sodium azide.
   12. Resuspend the pellet in 25 mL of 2% SDS 20 mM sodium phosphate pH 6.5.
   13. Boil for 1 h in a steamer or the round bottom flask with water-cooled condenser (section 1.2.2).
       NOTE: The second SDS boiling step removes all the remaining proteins and contaminants from the sacculi.
   14. Repeat the sacculi wash (section 1.2.6) with ~50 mL room temperature double distilled water (ddH₂O) until the SDS concentration is ~0.001%.
   15. Resuspend the pellet in a sufficient quantity of ddH₂O to suspend sacculi, as well as wash the container (e.g., 25 mL) and freeze overnight at -80 °C. The volume can vary as the sample will be lyophilized in the next step, although, smaller volumes require less time to lyophilize.
   16. The next day, lyophilize the suspension and store at room temperature.
   17. Measure the quantity of lyophilized sacculi obtained on an analytical balance.
   18. Dilute sacculi in ddH₂O to 10 mg/mL and briefly sonicate to break up clumps prior to further analysis.
3. Quantification of purified peptidoglycan
   1. Quantify the amount of purified sacculi from section 1.2.18 to ensure mass spec data is equalized during differential analysis (section 3.2). Follow the detailed methodology outlined in Reference¹⁵.
      NOTE: Purified sacculi (section 1.2.18) are broken down into individual sugar and amino acid components by acid hydrolysis. Individual components are separated and quantified by anion-exchange chromatography using pulsed-amperometric detection. Given the structure of PG (Figure 1), individual muropeptides are composed of a single MurNAc and a single GlcNAc residue. Therefore, quantifying the concentration of either residue represents the quantity of muropeptides 1:1 within the sample. MurNAc is preferred due to the clean peak separation from other PG components during chromatography¹⁶.

2. Mass spectrometry data acquisition

1. Preparation of muropeptides for mass spectrometry
   1. Supplement 800 μg of purified sacculi with 100 μg/mL mutanolysin, 100 mM ammonium acetate pH 5.5, and 50 mM magnesium chloride in a 100 μL reaction.
   2. Digest at 37 °C overnight.
   3. Add 1:1 volume 0.5 M borate buffer pH 9.0 and supplement with ~10 mg/mL sodium borohydride (NaBH₄).
      NOTE: Mutarotation of cyclic sugars between α and β anomeric forms will cause multiple peak formations during the liquid chromatography separation of the muropeptides. The treatment with NaBH₄ eliminates interconversion between the two forms by reducing MurNAc into muramitol¹¹. The treatment does not reduce 1,6-anhydro MurNAc or 1,4-linked sugar residues.
      CAUTION: The reaction of NaBH₄ produces small quantities of hydrogen gas. The NaBH₄ reaction will create bubbles and microfuge tubes should be kept open to allow gas to escape.
   4. Incubate the sample at room temperature for ~20–30 min.
   5. Centrifuge briefly to settle the sample in the microfuge tube and remove bubbles.
   6. Adjust pH to <4.0 using 1:5 phosphoric acid, added in 5 μL increments. Test pH using litmus pH test strips.
   7. Centrifuge at ~17,000 x g for 1 min to sediment any remaining insoluble material.
   8. Filter using 0.2 µm microcentrifuge filters.
   9. Samples are centrifuged for 10 min at 30,000 x g prior to injection into LC-MS to ensure any particulates are not injected into MS.
2. Setup of LC-MS
   1. Attach a C18 superficially porous particle column (100 mm x 2.1 mm, pore size <3 µm) to a Quadrupole-Time of Flight (QTOF) mass spectrometer with a minimal four decimal point m/z detection accuracy.
   2. Perform liquid chromatography separation of muropeptides
      NOTE: Each biological triplicate (section 1.1.1) should be run through the LC-MS (section 2.2.2 through 2.2.3) three times (technical triplicate). Therefore, each tested condition will have a total of nine LC-MS data files. Data acquisition was performed using commercially available software (see Table of Materials). However, acquisition software should be chosen based on the MS machinery. The following represents a guide for setting up the MS with parameters specific for running this protocol. For a detailed description, please refer to the manufacturer’s manual.
      1. For chromatographic separation, prepare the following solvents 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B).
      2. Set up a method for chromatographic separation using the following parameters.
      3. Set the flowrate to 0.4 mL/min.
      4. Condition the column for 10 min at 2% B (~24 column volumes).
      5. Using an autosampler, inject 10 µL of sample from section 2.1.9.
         NOTE: Run one initial sample through the LC-MS protocol prior to beginning data collection. This run is not used for data but to increase retention time reproducibility for all subsequent runs. The reproducibility of the retention time is required during spectral processing (section 3.1) for the accurate identification and grouping of mass-to-charge ratio (m/z) peaks.
      6. Separate muropeptides using 2% B for 5 min (~12 column volumes), then increasing it to 15% B over 13 min (~30 column volumes), further increasing it to 50% B over 10 min (~24 column volumes), and finally increasing it to 98% B over 2 min (~5 column volumes).
         NOTE: Discard (send to waste) the first 2 min and last 5 min of the gradient.
      7. Finish with a column wash at 98% B for 6 min (~14 column volumes) and 20 min (~47 column volumes) re-equilibration.
   3. Perform mass spectrometry detection of muropeptides
      1. Calibrate the mass axis in positive mode using a tuning mix in acetonitrile containing LC-MS reference mass standards following MS manufacturer’s instructions.
         NOTE: The MS is tuned prior to the beginning of the chromatographic runs (section 2.2.2.1).
      2. Set up a method for MS data acquisition using the following parameters.
         NOTE: MS and MS/MS data are collected (sections 2.2.3.2 to 2.2.3.6) simultaneously with the chromatographic separation of the muropeptides (sections 2.2.2.4 and 2.2.2.5). Both chromatographic and MS parameters (sections 2.2.2.2 and 2.2.3.2) are a single method that is added during the setup of a worklist for running multiple samples in sequence.
      3. Set the electrospray capillary voltage at 4.0 kV and the drying gas temperature at 350° C with a flow rate of 13 L/min.
         NOTE: Nitrogen (purity >99%) should be used as nebulizing, drying and collision gas during all mass spectrometry data collection.
      4. Set nebulizer pressure to 40 psi and set the fragmentor to 150 V.
      5. Set the nozzle, skimmer, and octapole RF voltages to 1000 V, 65 V, and 750 V, respectively.
      6. Set the scan range to 300–2,000 m/z in 4 GHz (extended dynamic range) positive ion mode.
      7. Set data collection using data dependent MS/MS acquisition with an MS and MS/MS scan rate of 1 spectra/second. Select five precursor mass per cycle, in the order of singly, doubly, and triply charged.
      8. Set MS/MS fragmentation collision energies to 15, 20 and 30 eV.

3. Differential analysis of muropeptide abundance

1. LC-MS chromatogram spectral processing
   NOTE: Recursive feature extraction was performed using commercially available software (see Table of Materials). Other feature extraction software can be used. However, other software may require additional manual data processing to accomplish the highly robust recursive extraction. Various software uses the terminology feature, entity, and compound almost interchangeably. For PG analysis, all refer to the identification of the LC-MS ion chromatogram representative of an individual muropeptide (e.g., Figure 3). During spectral processing (section 3.1), a feature represents the multiple m/z peaks that encompass the multiple possible ion species of a single muropeptide that are grouped together under a single compound label.
   1. Under File, start a new project.
   2. Add the LC-MS QTOF data files and assign individual data files to an experimental condition / group, e.g., two different growth conditions.
   3. Run the data processing wizard Batch recursive feature extraction (small molecules/peptides) and set the data processing filters to match the parameters of the LC-MS conditions and instrumentation to accurately identify, group and verify m/z peaks representing individual muropeptides. From the chromatogram, examine the retention time drift and variation of m/z of known similar peaks to set initial filter parameters.
      NOTE: Recursive feature extraction uses an initial untargeted molecular feature extraction algorithm to identify and align chromatogram features (m/z peaks) across all the data files. Once created, these features are used to reassess the original data files (recursive) with a targeted molecular feature extraction algorithm to improve the reliability and accuracy of the identified features. It is best to set the initial untargeted extraction with a narrow detection window and use broader detection parameters for the recursive extraction to identify peaks in all data files that may be missing in the initial extraction. Running the recursive feature extraction can take hours to complete depending on the number of samples, the complexity of the data, and the computer hardware present
   4. Review the feature extraction results. If a significant number of features failed to align in a group, adjust the recursive filtering parameters to broaden/restrict the detection window as required. To accomplish this, inspect the chromatogram and isotopic profile of each extracted feature to ensure feature detection was accomplished similarly across all data files. Also, for each identified feature within a data file, examine the score, any warnings and whether the feature passed the chosen filter parameters.
      NOTE: Care must be taken to keep detection windows narrow enough that distinct features are not mistakenly grouped together. This is often noted by disparate isotopic profiles between data files, or significant m/z / retention time variations. Conversely, if there are multiple features with similar m/z and retention times, it is possible that the filter parameters were too stringent resulting in one MS peak being split into two features. Therefore, run feature extraction (section 3.1.3) again with adjusted retention time filter parameters to allow better grouping of these features. Visualizing the lowest abundance peaks will indicate whether the filters used (section 3.1.3) accurately identified peaks above background noise. If the lowest abundance peaks appear similar to the background, rerun the feature extraction with new background filter parameters.
   5. Export data as a compound exchange file (.cef) (a format compatible with the statistical software program), or a column separated file (.csv) which contains the mass, retention time, and abundance of each feature for each sample.
2. Differential analysis of spectral features
   NOTE: Differential analysis was performed using commercially available software (see Table of Materials). Other bioinformatic software can be used.
   1. Open the program and when instructed start a new project.
   2. Follow the instructions for data import and data analysis. During data import, upload the feature extracted files (section 3.1). During data analysis, choose significance and fold change for differential analysis and select to baseline data to the median intensity across all the data files. Do not set any data filters (if this was done in the previous spectra processing step (section 3.1)) as applying filters again would negate the robust recursive feature extraction. However, similar to feature extraction, differential analysis must be aligned based on mass (m/z) and retention time due to drift in the LC-MS data collection. Use the parameters determined in feature extraction (section 3.1).
      NOTE: Normalize between data files using the muropeptide quantitation (section 1.3) to ensure variations are due to experimental parameters and not due to variations in sacculi purification (section 1.2). Use the external scaler option to adjust each data file for differences in sample MurNAc concentration.
   3. Once the analysis is complete, examine the resulting graphical and statistical analyses to identify muropeptides that demonstrate a significant abundance change between the tested experimental conditions.
   4. Under the project navigator, right-click on the various analyses and choose an export option to save feature details as a column separated (.csv) data file that contains the m/z, retention time, raw and normalized intensity values, p-value, FDR, and fold changes for each feature. Multiple analyses must be saved to obtain all the relevant data.
   5. Export a second .csv file containing only the muropeptides that have surpassed the statistical analyses including p-value <0.05 and fold change >2.
3. Annotating muropeptide identity to spectral features
   NOTE: Each identified feature must be assigned a predicted muropeptide structure based on the m/z and this annotation confirmed by examining the MS/MS fragmentation. After confirming the annotations, it may be necessary to perform and refine the differential analysis (section 3.2).
   1. Within the differential analysis software, under results interpretation, select ID browser. Add a library of expected muropeptide structures and select similar parameters as used previously (section 3.1.3). This will produce a predicted muropeptide annotation for each identified feature. A library of muropeptide structures can be produced using the m/z for predicted muropeptide structures and MS database software (see Table of Materials). However, a library of the m/z of >6,000 possible muropeptides can be found in Reference¹².
   2. Select the predicted muropeptide annotation based on the matching score and the biological relevance of the predicted muropeptide, i.e., choose the most likely muropeptide to be present in the biological sample.
   3. Manually confirm the predicted muropeptide annotation by comparing the m/z peaks of the MS/MS chromatogram to the predicted m/z of all possible fragmentations of a known muropeptide structure (e.g., Figure 4).
      1. View the MS and MS/MS data using a chromatogram-viewing program (see Table of Materials, Figure 4).
      2. Draw the predicted muropeptide structure using a molecular editor (chemical structure drawing program) (see Table of Materials, Figure 4, gray inset). Use the mass fragmentation tool to show the m/z of MS fragments when each bond is broken either individually or in combination.
         NOTE: Depending on the fragmentation energy used for MS/MS, fragmentation can happen at any bond in the muropeptide structure. However, some bonds are more easily / frequently fragmented at lower energy levels. For example, fragmentation in the peptide sidechain occurs most often at the amide bond between amino acids. While assessing the fragmentation, it is important to note that GlcNAc residues are very easily fragmented from the muropeptide. Therefore, fragmentation of the known muropeptide structure should be assessed with and without GlcNAc. Due to the in-source fragmentation of the GlcNAc, several features extracted in spectral processing (section 3.1) may represent a single muropeptide structure. If found, these features should be merged and the differential analysis reassessed.
      3. Compare all the possible fragmentations of the muropeptide structure that was determined (section 3.3.3.2) to the MS/MS chromatogram (section 3.3.3.1). To confirm the muropeptide annotation, the m/z peaks of multiple fragments should be found in the MS/MS chromatogram with a very minimal m/z alignment window (Figure 4).
      4. In order to elucidate muropeptide identity in the case of ambiguous MS/MS fragmentation, repeat sections 2.2.2 through 2.2.3 with samples (section 2.1.9) for additional MS/MS data acquisition incorporating a preferred precursor list of m/z and retention time with additional MS/MS fragmentation collision energies.
   4. For co-eluting entities that were annotated as the same muropeptide, run the differential analysis (section 3.2.2) again and merge the extracted features.
4. Assessing global changes in muropeptide modifications
   1. Edit the .csv file (section 3.2.4) of the statistically significant high fold change muropeptides to include a single column for each muropeptide modification. Populate this column with a designation for each muropeptide annotated (section 3.3) (e.g., acetylated versus de-N-acetylated GlcNAc or MurNAc).
   2. Upload the modified .csv file into Perseus^22,23. Import the normalized intensity values into the Main import box and import the modification designation into the Categorical import box.
   3. Under Annotate Rows, click on Categorically Annotate Rows and add datafiles to each experimental parameter.
   4. Under test, click on two sample tests to perform a student’s t-test (p-value < 0.05, FDR < 0.05, s0 = 1).
   5. Click on 1D to perform 1D annotation^22,23. A 1D annotation FDR < 0.05 indicates a significant abundance change for the muropeptide modification between the tested experimental parameters. Setting the Threshold value (s0) = 1 will display the 1D annotation FDR scores for all the muropeptides.
   6. Within a graphing software (see Table of Materials), produce a heat map of the abundance fold change for each muropeptide modification and show the 1D annotation score to demonstrate significance (Figure 5B). The fold change of each muropeptide modification can be produced in Microsoft Excel using the raw intensities of all individual muropeptides that contain the modification.