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Jan 2020

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Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography
革兰阴性菌肽聚糖的超高效液相色谱法分析   

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Abstract

Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry (MS) of the UPLC resolved muropeptides, thus facilitating their identification. This method improves our capability to perform high throughput analysis to better understand the cell-wall biology.

Keywords: UPLC (超高效液相色谱), Cell Wall (细胞壁), Peptidoglycan (肽聚糖), Muropeptide (胞壁肽), Muramidase (溶菌酶), Crosslink (交联)

Background

Bacteria are surrounded by a peptidoglycan (PG) cell wall that, in addition to a structural role, conveys cell shape and protects bacteria from external damage, acting as a barrier against biological, chemical and physical stresses. The murein sacculus or PG is the major component of the cell wall. Gram-negative bacteria present a monolayer in the periplasmic space (Gan et al., 2008), while it constitutes a thick mesh work with several pilled and crosslinked layers in Gram-positive bacteria, (Pasquina-Lemonche et al., 2020). The cell wall is a three-dimensional meshwork of crosslinked glycan strands that encloses the cell body (Glauner et al., 1988; Typas et al., 2011; Egan et al., 2020). The canonical monomeric subunit consists of the disaccharide pentapeptide GlcNAc-(β1-4)-MurNAc-L-Ala-D-Glu-(γ)-(di-amino acid)-D-Ala-D-Ala, where meso-diaminopimelic acid and L-lysine are the most prevalent di-amino acids (Glauner et al., 1988; Vollmer et al., 2008). These monomers are converted into linear polymers by means of MurNAc-(β1-4)-GlcNAc glycosidic bonds and can be crosslinked by peptide chains.

The cell wall is subject to various enzymatic modifications that affect the chemical properties of its subunits, their relative abundance, and the structure of the cell wall (Vollmer et al., 2008). A detailed knowledge of the muropeptide composition under particular conditions is key to understanding to what extent PG variations influence bacterial adaptation to environmental challenges, resistance to antibacterial agents, immune-modulatory activity and toxin release and signaling (Alvarez et al., 2014; Cava and de Pedro, 2014; Yadav et al., 2018). Our method allows us to study the effect of genetic perturbations on cell-wall composition in a high-throughput manner. Furthermore, combining high-throughput PG analysis with genetic perturbations, we can explore the functions of different cell-wall-modifying enzymes. In fact, we have recently studied how class-A Penicillin-Binding Proteins (aPBPs) contribute to cell-wall integrity in Escherichia coli (Vigouroux et al., 2020). By combining PG analysis, morphology analysis and single-molecule tracking, we have demonstrated that the major aPBP PBP1b contributes to cell-wall integrity by repairing cell-wall defects, while the Rod complex governs rod-like cell shape.

Here we describe in detail the procedure for Gram-negative bacteria sacculi isolation, muramidase digestion and muropeptide separation by liquid chromatography. We also provide some general instructions on compositional and structural analysis. The isolation of PG relies on the insolubility of the sacculi in SDS, which allows a relatively easy method to obtain high amounts of PG, and on the availability of specific enzymes (muramidases or lysozymes) that split the MurNAc-(β1-4)-GlcNAc glycosidic bonds which hold the structure together and disassemble it into its individual subunits. The next step requires the use of sensitive and reliable methods that permit the resolution, identification and quantification of the different PG subunits. Since the 80’s, HPLC has been traditionally used for this purpose (Glauner et al., 1988). While this technology was a revolution at the time and revealed an unexpected complexity in PG structure and composition, it has been used essentially unchanged for more than 30 years. However, there are three critical limitations: i) the requirement for inorganic buffers, incompatible with mass spectrometry in-line analysis that would facilitate identification of subunits; ii) the very low sample through-put, which requires a few days for sample preparation and several hours of HPLC run time per sample; and iii) the requirement for large sample volumes (injection volumes of 100-500 µl), due to the relatively low sensitivity of the chromatographic systems. The introduction of UPLC replacing its predecessor HPLC permits to dramatically reduce sample size (100x), while increasing the speed (20x), without compromising the quality of the data. The new systems allow the use of new and improved materials for reverse phase chromatography, including stationary phases with a very small particle size (in the range of 2 µm) that withstand very high pressures, increasing resolution, speed and sensitivity, which are essential requisites for high throughput analysis.

We have developed a new transformative approach that circumvents the limitations of the already available methods (e.g., Desmarais et al., 2013; Kühner et al., 2014). Our protocol i) can be easily adapted for the more frequently available UPLC machines by anyone with a basic knowledge of UPLC techniques; ii) has cut down sample preparation and run times dramatically, allowing for the processing and analysis of large numbers of PG samples per day; iii) is MS-compatible, samples can be collected and subjected to MS analysis without prior desalting steps or can even be analyzed in MS-systems coupled to the UPLC for a faster muropeptide identification, thus revealing a much higher PG chemical complexity than previously anticipated (i.e., identification of minor muropeptides); iv) and requires much less of sample, which is particularly important for in vivo samples (low sample size amount).

Materials and Reagents

  1. Disposable pipette tips (VWR, catalog numbers: 613-1083 , 613-1079, 613-1077)
  2. 1.5 ml microtubes (Eppendorf, catalog number: 00 30120086 )
  3. 2 ml microtubes (Eppendorf, catalog number: 00 30120094 )
  4. 15 ml conical test tubes (Sarstedt, catalog number: 62.554.502 )
  5. 50 ml conical test tubes (Sarstedt, catalog number: 62.547.205 )
  6. Thickwall polycarbonate ultracentrifuge 3.5 ml tubes, 13 x 51 mm (Beckman, catalog number: 349622 )
  7. pH-indicator strips: pH range 5.0-10.0 and pH range 0.0-6.0 (Merck, catalog numbers: 109531 , 109533)
  8. 96-well V-shaped-bottom microplates (Corning, Falcon, catalog number: 353263 )
  9. Pierceable adhesive seal for microplates (Waters, catalog number: 186006336 )
  10. Analytical column: Kinetex C18 UPLC Column 1.7 µm particle size, 100 Å pore size, 150 x 2.1 mm (Phenomenex, catalog number: 00F-4475-AN )
  11. Precolumn filter or guard column: SecurityGuard ULTRA Cartridges C18 for 2.1 mm ID columns (Phenomenex, catalog number: AJ0-8782 )
  12. Precolumn holder: SecurityGuard ULTRA Holder, for UHPLC Columns 2.1 (Phenomenex, catalog number: AJ0-9000 )
  13. MilliQ water
  14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653-250G )
  15. Potassium chloride (KCl) ((Sigma-Aldrich, catalog number: P9333-500G )
  16. Sodium phosphate monobasic dihydrate (NaH2PO4•2H2O) (Sigma-Aldrich, catalog number: 71505-250G )
  17. Sodium phosphate dibasic dihydrate (Na2HPO4•2H2O) (Sigma-Aldrich, catalog number: 71643-250G )
  18. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655-100G )
  19. Trizma base (Tris(hydroxymethyl)aminomethane) (Sigma-Aldrich, catalog number: T6791-100G )
  20. Ortho-phosphoric acid 85% (H3PO4) (Merck, catalog number: 1005731000 )
  21. Hydrochloric acid fuming 37% (HCl) (Merck, catalog number: 1003171000 )
  22. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L6026-50G )
  23. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768-500G )
  24. Sodium hydroxide pellets (NaOH) (Merck, catalog number: 1064821000 )
  25. Sodium borohydride (NaBH4) (Merck, catalog number: 1063710100 )
  26. Formic acid (CH2O2) (Merck, catalog number: 5330020050 )
  27. Acetonitrile (CH3CN) (Merck, catalog number: 1000291000 )
  28. Proteinase K 20 mg/ml (Thermo Scientific, catalog number: EO0491 ), store enzyme at -20 °C
  29. Mutanolysin from Streptomyces globisporus ATCC 21553 (Sigma-Aldrich, catalog number: SAE0092-10KU). Resuspend in 2.5 ml digestion buffer (stock concentration ~1 mg/ml) and store enzyme at -20 °C
  30. Phosphate buffer saline (PBS) (see Recipes)
  31. Lysis buffer [SDS 5% (w/v)] (see Recipes)
  32. Tris HCl 1 M pH 8 buffer (see Recipes)
  33. Tris HCl 100 mM pH 8 buffer (see Recipes)
  34. SDS 10% (w/v) solution (see Recipes)
  35. Digestion buffer (see Recipes)
  36. NaOH 2 M solution (see Recipes)
  37. Borate buffer (see Recipes)
  38. NaBH4 solution (see Recipes)
  39. Ortho-phosphoric acid 25% (v/v) solution (see Recipes)
  40. Organic buffer A (see Recipes)
  41. Organic buffer B (see Recipes)

Equipment

  1. Pipettes (Gilson, catalog numbers: F144563 , F144565 , F144566 )
  2. Glassware: bottles, measurement cylinders, beakers
  3. pH-meter (VWR, catalog number: 662-1422 )
  4. Hot plate stirrer (VWR, catalog number: 97042-598 )
  5. 12 x 6 mm magnetic stirring bars (VWR, catalog number: 442-4501 )
  6. Centrifuge for microtubes (Eppendorf, Centrifuge 5418, catalog number: 5418000017 )
  7. Centrifuge for 15/50 ml conical tubes (Eppendorf, Centrifuge 5920 R, catalog number: 5948000914 )
  8. Beckman Optima MAX-TL Ultracentrifuge (Beckman, catalog number: A95761 )
  9. Ultracentrifuge TLA-100.3 rotor (Beckman, catalog number: 349490 )
  10. Vacuum pump
  11. Laminar flow cabinet
  12. Acquity UPLC system (Waters) or similar (e.g., Agilent)

Software

  1. Waters Empower 3, build 3471 (Waters, https://www.waters.com/waters/en_US/Empower-3-Chromatography-Data-Software/nav.htm?locale=en_US&cid=10190669)
  2. Microsoft Excel Version 2004 (Microsoft, Office 365, https://www.microsoft.com/en-us/microsoft-365/excel?rtc=1)
  3. Prism 8 Version 8.0.2 (GraphPad, https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Cell lysate preparation
    1. Grow cultures to the desired optical density in the appropriate culture medium for the bacteria. Record the culture optical density for normalization. For optimal sample preparation, aim for 1010 bacteria or higher (e.g., 10 ml at 1 unit OD600, 50 ml at 0.2 units OD600). Samples can be scaled up but require more reagents and are more time-consuming.
      For statistical analysis, prepare and process samples in triplicates.
    2. Transfer the cultures to 50 ml conical tubes and harvest the cells at 3,000 x g for 15 min. Remove the supernatant and resuspend the pellet in 1.5 ml of its own media. Alternatively, pellets can be resuspended in PBS or Tris HCl buffers. Final volume is not critical, but lower volumes are preferred to shorten the number of washes required later (Figure 1A).
      Note: Resuspension media containing potassium (e.g., M63 minimal medium) can interfere with sacculi preparation as potassium salts react with SDS producing insoluble KDS.
    3. Transfer the samples to 15 ml conical tubes. The tubes must be suitable for boiling. Place a small stirring bar (magnet) per tube (Figure 1A).


      Figure 1. Pipeline for peptidoglycan sample preparation. A. Lysate preparation: bacterial cultures are collected and SDS is added to the resuspended pellets, which are boiled and stirred to facilitate solubilization of all cell components except for the peptidoglycan sacculus. B. Sacculi are washed repeatedly with water and submitted to enzymatic treatment. Released muropeptides in the supernatant are injected in a chromatographic system. C. Mobile phase gradient applied during UPLC separation. D. Steps of the gradient separation of muropeptides by UPLC.

    4. Add 1.5 ml lysis buffer and place the tubes on a beaker with boiling water on a magnetic hot stirrer plate. Boil the samples for 30 min-2 h, then switch off the hot plate and let the lysate stir overnight. Cells will immediately lyse upon boiling in SDS, but longer stirring times are recommended for shearing the DNA (Figure 1A).
    5. Boiled lysates can be stored at room temperature for long time. Make sure the lid is tightly closed to avoid desiccation.

  2. Isolation of Gram-negative bacteria peptidoglycan
    1. To completely remove SDS, perform several washes with water by centrifugation. Spin down the samples 10 min and 150,000 x g using 3 ml polycarbonate ultracentrifuge tubes. Use 20 °C or higher since SDS precipitates at low temperature. Fill the tubes with 3 ml lysate. Make sure the rotor is properly balanced. After centrifugation, all soluble compounds will remain in the supernatant. The resulting pellet is usually transparent and hard to distinguish. It is recommended to mark one side of the tube and use this as reference to locate the pellet (Figure 1B).
      Note: If during the washes a white precipitate (likely insoluble KDS) is observed, use Tris HCl 100 mM pH 8.0 buffer instead of water.
    2. Carefully discard the supernatant with a vacuum pump without removing the pellet. Resuspend the pellet in 900 µl MilliQ water and check for the presence of SDS by assessing the production of bubbles. If needed, add 2 ml MilliQ water, mix and centrifuge again. Repeat this wash step until SDS is completely removed (no bubbles are formed after the pellet is resuspended in water, typically 2-4 washes).
    3. To remove Braun’s lipoprotein or other PG-associated proteins, treat the washed sacculi with a protease. This step is optional, but highly recommended. Transfer the resuspended pellet (~900 µl) to 2 ml microtubes. Add 100 µl Tris HCl 1 M pH 8 buffer and 1 µl proteinase K 20 mg/ml. Incubate samples at 37 °C during 1 h. Stop the reaction by adding 110 µl SDS 10% (w/v) and boil for 5 min in water.
      Note: Proteinase K is a broad-spectrum serine protease used for removal of PG bound proteins such as Braun’s lipoprotein. The enzyme retains activity in SDS 1% (w/v). Some bacteria do not require Proteinase K treatment, but it is recommended for removal of other PG-associated proteins. Other proteases as chymotrypsin (EC 3.4.21.1) or trypsin (EC 3.4.21.4) can also be used.
    4. Let the sample cool down before transferring to the ultracentrifuge tubes again. Wash the Proteinase K digested sacculi by adding 2 ml MilliQ water. Mix and ultracentrifuge as described before. Carefully discard the supernatant and, after total removal of SDS, resuspend the pellet in 50-100 µl digestion buffer. The final volume is critical, since soluble muropeptides will remain in the soluble fraction after the muramidase digestion. Washed sacculi should be stored at 4 °C to avoid degradation or contamination. Storing the samples at -20 °C is not recommended since sacculi can break due to freeze/thaw processes.

  3. Muramidase digestion
    1. Transfer resuspended sacculi (50-100 µl) to 1.5 ml microtubes.
    2. Add 2 µl mutanolysin 1 mg/ml and let the reaction work 2-16 h at 37 °C. The reaction is almost complete after 2 h, but to ensure total digestion, let it proceed for longer times. 
    3. To inactivate the enzyme, boil the samples for 5 min. Do not add any detergent to the sample.
    4. Centrifuge 15 min at room temperature and 20,000 x g in a benchtop centrifuge and transfer the supernatant to new 1.5 ml microtubes. This supernatant contains the soluble muropeptides released by the mutanolysin (Figure 1B). These samples are very stable and can be stored at 4 or -20 °C.

  4. Sample reduction
    Note: Sample reduction is optional but highly recommended. Digestion with mutanolysin produces a terminal reducing NAM. Sample reduction avoids the different anomeric configurations and the consequent appearance of two to four peaks per muropeptide in the PG profile, as the NAM is reduced to muramitol. Perform this procedure in a laminar flow cabinet. If no reduction is performed, adjust the sample pH to 2.0-4.0 with diluted orthophosphoric acid instead (0.25%, v/v).
    1. Add borate buffer to the sample to adjust pH to 8.5-9.0. For a 100 µl reaction, 15-20 µl borate buffer are typically used. Check pH using indicator strips: take 0.5 µl sample and drop on the indicator strip. Check the color/pH on the reference table. Do not leave samples at high pH for too long, since it leads to alkaline β-elimination (Tipper, 1968).
    2. Add 20 µl freshly prepared NaBH4 2 M and let the sample reduce at room temperature for 30 min. This reaction produces H2 and bubbles are accumulated, do not close the tube lids to prevent gas accumulation.
    3. Adjust sample pH to 2.0-4.0 with orthophosphoric acid 25% (v/v). Addition of the acid will cause a violent reaction and sudden bubble formation. First add 4 µl and check the pH with the appropriate indicator strip as indicated before. If needed, carefully add acid µl by µl to ensure the sample reaches the desired pH.
    4. Spin the tubes for 1 min at maximum speed to ensure maximum sample recovery. Transfer the samples to a 96-well V-shaped-bottom microplate. Seal the plate using a pierceable adhesive seal. Samples can be stored long-term at -20 °C. Insoluble precipitates might form and if observed, filter the samples prior to injection in the UPLC.

  5. UPLC separation
    Note: settings will depend on the software, model and manufacturer of the UPLC. For muropeptide profiling, reverse-phase (RP) columns, typically C18-bonded silicas that are able to retain and separate medium-polar and non-polar metabolites provide a good separation pattern. For RPLC, maximum retention of analytes is ensured by loading samples onto the column using solvents of low eluotropic strength (i.e., composed mainly or entirely of water). Elution of retained metabolites is accomplished using a gradient of increasing acetonitrile content.
    1. Set the column temperature to 45 °C.
    2. Prepare mobile phases and refill bottles A and B with organic buffer A [formic acid 0.1% (v/v)] and organic buffer B [formic acid 0.1% (v/v) in acetonitrile] respectively. Purge pumps and tubes according to the UPLC system instructions.
    3. Equilibrate the column with organic buffer A, flow 0.25 ml/min until pressure is stabilized.
    4. Using the system auto-sampler, inject 10 µl sample.
    5. Perform the LC run using the gradient described in Figures 1C and 1D and measure absorbance at 204 nm.
    6. Run a blank injection to monitor and subtract the baseline.

  6. Troubleshooting
    1. Carefully control parameters such as flow rate and column temperature following the manufacturers indications to ensure repeatability of the separations. Typically, ~2,000 samples can be injected in a chromatographic column before separation efficiency is lost, peaks degrade and retention times change. If the chromatogram shows poor peak shapes, this is indicative of column degradation or sample overloading. To solve this problem, dilute the sample or improve sample preparation. If the problem persists, consider cleaning or replacing the column.
    2. The injection volume can be modified depending on the sample concentration:
      1. For concentrated samples (e.g., PG from large starting cultures), inject less volume, ensuring the system is working within its detection limits.
      2. If there are no or few peaks or the signal is too low, increase the injection, concentrate the sample using a Speedvac concentrator or prepare new sample by resuspending the washed sacculi in a lower volume prior to mutanolysin digestion.
    3. Changes in the retention time can be due to improper sample pH or presence of detergent (SDS) in the sample. When sample pH is greater than 5, it results in shifted chromatograms. Adjust pH with orthophosphoric acid 25% (v/v) and rerun the sample. If there is detergent in the sample, either prepare new sample increasing the amount of washing steps or wash the column after each run to get rid of the retained detergent.
    4. Improper sample reduction results in profiles with altered retention times. Repeat the sample preparation adding an excess of NaBH4.
    5. Contamination with other components or short re-equilibration time between runs also contribute to the appearance of ghost peaks.
    6. When sample concentration is low, the baseline drift and noise become more evident. Run a blank injection with water and subtract the baseline during the data processing.

Data analysis

Prepare and run samples in triplicates for statistical analysis.
For optimal comparison conditions, use the same volumes, washes and treatments for all samples.

  1. Peptidoglycan profile
    1. Extract the raw data: retention time (min) and absorbance at 204 nm (arbitrary units).
    2. For baseline correction, subtract the data of a blank injection of water (Figure 2A).
    3. Define the chromatographic processing regions removing not useful data, typically the injection front and the wash at the end of the run (datapoints ≤ 2-3 min, datapoints ≥ 12-13 min) (Figure 2A).
    4. Represent the chromatogram by plotting absorbance at 204 nm (arbitrary units) against retention time (min) (Figure 2A).


      Figure 2. Schematics for analysis of peptidoglycan data. A. Transformation of the raw data for optimal absorbance versus time representation. Subtract a blank injection for baseline correction and trim the datapoints to be shown. B. Example of a simple peptidoglycan chromatogram. C. Formulas employed for the quantifications of muropeptide relative molar abundances and crosslink. D. Table showing the muropeptide abundances in the example profile. E. Peptidoglycan features calculated from the table of muropeptide abundances.

    5. Identify the muropeptides by comparison to a known reference sample run in the same chromatographic system. Unknown or novel peaks can only be identified by MS analysis.

  2. Relative total PG amount
    1. For normalization, calculate the relative cell amounts by dividing the recorded optical densities at the time of harvesting by the average optical density of the control samples.
    2. Calculate the total area of the chromatogram by summing all the intensity values in the time range of interest (min 2-3 to min 12-13).
    3. Normalize the total area by dividing the total area of each sample by its relative cell amount. 
    4. Divide the normalized total area by the average value of the control samples, and then multiply the result by 100 to calculate the relative percent.
    5. Perform an unpaired t-test analysis to determine the statistical significance of the results.

  3. Muropeptide quantifications
    1. For quantifications, calculate the area of each peak by integration using the appropriate software (e.g., UPLC manufacturers software, MATLAB) (Figure 2B).
    2. To calculate the peak molar abundance, divide each area by the muropeptide molar ratio: for simplification, 1 if it is a monomer, 2 if it is a dimer, 3 if it is a trimer, … (Figure 2C).
    3. Determine the relative molar abundances for each peak: divide the molar abundance of every peak by the total of the chromatogram (sum of all individual molar abundances) and multiply the result by 100 to calculate the relative percentage (Figure 2C).
    4. Perform an unpaired t-test analysis to determine the statistical significance of the results.
    5. Represent the results as a muropeptide table that typically contains retention time and relative molar abundance for all detected muropeptides (Figure 2D).

  4. Calculation of peptidoglycan main features
    1. Monomers (%): sum the relative molar abundances of all monomeric muropeptides.
    2. Dimers (%): sum the relative molar abundances of all dimeric muropeptides.
    3. Trimers (%): sum the relative molar abundances of all trimeric muropeptides.
    4. Crosslink (%): calculate the percentage of crosslink as Dimers + (Trimers × 2). There is a crosslink in a dimer, 2 in a trimer (Figure 2C).
    5. Anhydro (%): sum the relative molar abundances of all anhydro muropeptides (muropeptides with (1-6anhydro)MurNAc).
    6. Chain length (glycan units): calculate the average glycan chain length by dividing 100 by the percentage of anhydro muropeptides.
    7. Perform an unpaired t-test analysis to determine the statistical significance of the results.
    8. Represent the results as a PG feature table (Figure 2D).

    A real example of PG analysis is shown in Figure 3.


    Figure 3. Peptidoglycan analysis of Escherichia coli samples with different repression levels of PBP1b and/or PBP1a. A. Representative UPLC profiles. Identified muropeptides are indicated. B. Relative PG amount (compared to AV84 5-5). C. Percentage of crosslink. Calculated as: dimers + (trimers × 2). D. Main PG features showing relative molar abundance of monomers, dimers and trimers, overall crosslink values, relative molar amount of anhydro muropeptides and estimated chain length. Statistical analysis: unpaired t-test. NS: not significant. Data represents mean ± SEM, 3 replicas. Data modified from Vigouroux et al., 2020.

Notes

  1. This protocol is not recommended for preparation of PG from most Gram-positive bacteria, since the existence of associated cell-wall polymers such as wall teichoic acids or lipoteichoic acids requires a more elaborated procedure for their removal (Mann et al., 2016).
  2. This protocol also works with lysed samples (e.g., treatment with an antibiotic such as D-cycloserine), as long as an ultracentrifugation step is implemented before sample collection for sacculi preparation:
    1. Spin down the samples at 4 °C and 200,000 x g for 30 min and discard the supernatant.
    2. Resuspend the pellet corresponding to the insoluble fraction in 1.5 ml supernatant or alternative media.
    3. Proceed with Step A3.

Recipes

Prepare all solutions with fresh MilliQ water. Use ultrapure water from a distillation or deionization unit with a resistance of 8 MΩ/cm at 25 °C. For the UPLC buffers, use only analytical grade reagents. Since pH is critical in most solutions used, make sure to gently stir the solutions and adjust the pH at a temperature as similar as possible to the working conditions. Solutions are stored at room temperature.


  1. Phosphate buffer saline (PBS)
    1. Dissolve 8 g NaCl, 0.2 g KCl, 1.77 g Na2HPO4·2H2O and 0.24 g KH2PO4 in 800 ml MilliQ water
    2. Stir until completely dissolved
    3. Adjust to pH 7.4 using HCl
    4. Adjust final volume to 1 L with MilliQ water
    5. Sterilize by autoclaving
  2. Lysis buffer [SDS 5% (w/v)]
    Dissolve 5 g SDS in 100 ml MilliQ water
    Note: Use protective gear since SDS powder may cause irritation of the respiratory tract.
  3. Tris HCl 1 M pH 8.0 buffer
    1. Dissolve 12.11 g Tris base in 80 ml MilliQ water
    2. Stir until completely dissolved
    3. Adjust to pH 8.0 using HCl
    4. Adjust final volume to 100 ml with MilliQ water
  4. Tris HCl 100 mM pH 8.0 buffer
    1. Dilute 10 ml of Tris HCl 1 M pH 8 buffer in 80 ml MilliQ water
    2. Adjust final volume to 100 ml with MilliQ water
  5. SDS 10% (w/v) solution
    Dissolve 10 g SDS in 100 ml MilliQ water
    Note: Use protective gear since SDS powder may cause irritation of the respiratory tract.
  6. Digestion buffer (50 mM phosphate buffer pH 4.9)
    1. Dissolve 887.6 mg NaH2PO4•2H2O and 9.5 mg Na2HPO4•2H2O in 80 ml MilliQ water
    2. Stir until completely dissolved
    3. Adjust pH to 4.9 with orthophosphoric acid 25% (v/v) solution to finely adjust pH
    4. Adjust volume to 100 ml with MilliQ water
  7. NaOH 2 M solution
    Dissolve 8 g NaOH in 100 ml MilliQ water and stir until completely dissolved
    Notes:
    1. This reaction is highly exothermic, safety measures must be taken.
    2. The NaOH solution is relatively unstable and its concentration can change over time, so for better accuracy, it is better prepared fresh.
  8. Borate buffer (borate buffer 0.5 M pH 9.0)
    1. Dissolve 3.1 g boric acid in 80 ml MilliQ water and stir until completely dissolved
    2. Adjust pH to 9.0 with NaOH 2 M solution
    3. Adjust final volume to 100 ml with MilliQ water
  9. NaBH4 solution (NaBH4 2 M)
    Dissolve 76 mg NaBH4 in 1 ml MilliQ water
    Note: Do not close any tube lids, since this reducing agent reacts with the solvents producing H2 in a violent reaction. This solution must be prepared fresh, do not store.
  10. Orthophosphoric acid 25% (v/v) solution
    1. Dilute 29.4 ml orthophosphoric acid 85% in 50 ml MilliQ water
    2. Stir until completely diluted
    3. Adjust final volume to 100 ml with MilliQ water
  11. Organic buffer A [formic acid 0.1% (v/v)]
    Dilute 1 ml HPLC grade formic acid in 1 l MilliQ water and mix
  12. Organic buffer B [formic acid 0.1% (v/v) in acetonitrile]
    Dilute 1 ml HPLC grade formic acid in 1 l acetonitrile and mix

Acknowledgments

This work reports in detail the sample preparation and analysis of peptidoglycan samples from Gram-negative bacteria. This work was supported by the Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Kempe Foundation and the Laboratory for Molecular Infection Medicine Sweden. A project grant from Stiftelsen Clas Groschinskys Minnesfond is also acknowledged. Work at the Institut Pasteur was supported by the European Research Council (ERC) under the Europe Union’s Horizon 2020 research and innovation program [Grant Agreement No. (679980)], the French Government’s Investissement d’Avenir program Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (ANR-10-LABX-62-IBEID), the Mairie de Paris ‘Emergence(s)’ program, and the Volkswagen Foundation. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. This protocol was derived from the E. coli PG analysis method published in our work (Vigouroux et al., 2020).

Competing interests

The authors declare no conflict of interest or competing interest.

References

  1. Alvarez, L., Espaillat, A., Hermoso, J. A., de Pedro, M. A. and Cava, F. (2014). Peptidoglycan remodeling by the coordinated action of multispecific enzymes. Microb Drug Resist 20(3): 190-198.
  2. Cava, F. and de Pedro, M. A. (2014). Peptidoglycan plasticity in bacteria: emerging variability of the murein sacculus and their associated biological functions. Curr Opin Microbiol 18: 46-53.
  3. Desmarais, S. M., De Pedro, M. A., Cava, F. and Huang, K. C. (2013). Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly. Mol Microbiol 89(1): 1-13. 
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简介

[摘要]细菌被保护性肽聚糖细胞壁包围。如果这种结构和涉及的酶是我们最成功的抗生素的首选靶标,那么确定其结构和化学复杂性便是最重要的。传统上,已经进行了高效液相色谱(HPLC)分析,但是这些方法在样品制备和色谱分离方面非常耗时。在这里我们描述了一种优化的制备方法 革兰氏阴性细菌肽聚糖及其随后的超高效液相色谱(UPLC)分析。在肽聚糖分析中使用UPLC可以大大减少所需的样品量和动手时间,此外,还可以对U PLC解析的多肽进行在线质谱(MS),从而有助于对其进行鉴定。这种方法提高了我们执行高通量分析以更好地了解细胞壁生物学的能力。

[背景]细菌由肽聚糖(PG)的细胞壁所包围,除了到结构的作用,传达细胞形状和保护细菌免受外部损害,作为抗生物,化学屏障iCal和物理应力。murein囊或PG是细胞壁的主要成分。革兰氏阴性细菌在周质空间中呈现单层(Gan等,2008),而它构成了一个厚的网状结构,在革兰氏阳性细菌中具有多个堆积和交联的层(Pasquina-Lemonche等,2020)。Ť他细胞壁是交联的聚糖链的三维网状结构包围所述电池主体(Glauner等人,1988; Typas 。等人,2011;伊根。等人,2020)。典型的单体亚基由二糖五肽GlcNAc-(β1-4)-MurNAc-L-Ala-D-Glu-(γ)-(二氨基酸)-D-Ala-D-Ala组成,其中内消旋-二氨基庚二酸氨基酸和L-赖氨酸是最普遍的二氨基酸(Glauner等,1988; Vollmer等,2008)。这些单体通过MurNAc-(β1-4)-GlcNAc糖苷键转化为线性聚合物,并且可以通过肽链交联。

细胞壁受到各种酶的修饰,这些修饰会影响其亚基的化学性质,其相对丰度以及细胞壁的结构(Vollmer等,2008)。特定条件下的muropeptide组合物的详细知识是关键是理解到什么程度PG变化影响细菌适应环境挑战,抗菌剂的抗性,免疫调节活性和毒素释放和信令(阿尔瓦雷斯等人,2014;卡瓦和de Pedro,2014; Yadav等,2018)。我们的方法使我们能够以高通量的方式研究遗传扰动对细胞壁组成的影响。此外,结合高通量PG分析和遗传扰动,我们可以探索不同的细胞壁修饰酶的功能。实际上,我们最近研究了A类青霉素结合蛋白(aPBP)如何对大肠杆菌的细胞壁完整性作出贡献(Vigouroux等,2020)。通过组合PG分析,形态分析和单分子跟踪,我们已经证明,主要aPBP PBP1b有助于小号通过修复细胞壁缺陷细胞壁完整性,而杆复杂共治棒状细胞的形状。

在这里,我们详细描述了通过液相色谱法对革兰氏阴性菌的葡萄球菌进行分离,muramidase消化和多肽分离的过程。我们还提供一些有关成分和结构分析的一般说明。PG的分离取决于SDS中囊囊的不溶性,这是一种相对容易的方法来获取大量PG,并且依赖于可分裂MurNAc-(β1-4)-的特定酶(村酰胺酶或溶菌酶)的可用性GlcNAc糖苷键将结构结合在一起并分解成单个亚基。下一步需要使用敏感和可靠的方法,以允许对不同的PG亚基进行解析,鉴定和定量。从80年代开始,HPLC就一直用于此目的(Glauner等,1988)。尽管这项技术在当时是一次革命,并且揭示了PG结构和组成中出乎意料的复杂性,但30多年来基本上没有变化。但是,存在三个关键局限性:i)对无机缓冲液的要求,与质谱在线分析不兼容,后者会有助于亚基的鉴定;ii)样品通量非常低,这需要几天的样品制备时间和每个样品几个小时的HPLC运行时间;iii)由于色谱系统的灵敏度相对较低,因此需要大量样品(进样量为100-500 µl)。引入UPLC替代其之前的HPLC可以在不影响数据质量的情况下,极大地减少样品量(100x),同时提高速度(20x)。新系统允许使用新的和改进的材料进行反相色谱分析,包括具有非常小的粒径(在2 µm范围内)的固定相,可以承受很高的压力,提高分离度,速度和灵敏度,这是必不可少的用于高通量分析。

我们已经开发出一种新的变革性方法,以克服现有方法的局限性(例如,Desmarais等,2013;Kühner等,2014)。具有UPLC技术基础知识的任何人都可以轻松地将我们的协议i)适应于更频繁使用的UPLC机器;ii)大大减少了样品制备和运行时间,从而每天可处理和分析大量PG样品;iii)与MS兼容,无需事先脱盐步骤就可以收集样品并进行MS分析,甚至可以在与UPLC联用的MS系统中进行分析以更快地鉴定多肽,从而揭示了比以前预期的PG化学复杂性高得多的方法(即鉴定较小的多肽);iv)并且需要更少的样品,这对于体内样品(低样品量)尤为重要。

关键字:超高效液相色谱, 细胞壁, 肽聚糖, 胞壁肽, 溶菌酶, 交联

材料和试剂
一次性移液器吸头(VWR,目录号:613-1083、613-1079、613-1077)
1.5毫升微管(Eppendorf,目录号:0030120086)
2 ml微型管(Eppendorf,目录号:0030120094)
15 ml锥形试管(Sarstedt,目录号:62.554.502)
50 ml锥形试管(Sarstedt,目录号:62.547.205)
厚壁聚碳酸酯超速离心机3.5 ml管,13 x 51 mm(Beckman,货号:349622)
pH指示条:pH范围5.0-10.0和pH范围0.0-6.0(Merck,目录号:109531,109533)
96孔V形底部微孔板(Corning,Falcon,货号:353263)
用于微孔板的可刺穿的粘性密封件(Waters,目录号:186006336)
分析柱:Kinetex C18 UPLC色谱柱粒径1.7 µm,孔径100Å,150 x 2.1 mm(Phenomenex,目录号:00F-4475-AN)
              柱前过滤器或保护柱:适用于2.1 mm ID柱的SecurityGuard ULTRA柱C18(Phenomenex,目录号:AJ0-8782)
前柱固定器:SecurityGuard ULTRA固定器,用于UHPLC色谱柱2.1(Phenomenex,目录号:AJ0-9000)
MilliQ水
氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653-250G)
氯化钾(KCl)((Sigma-Aldrich,目录号:P9333-500G)
磷酸二氢钠一水合物(NaH 2 PO 4 ·2H 2 O)(Sigma-Aldrich,目录号:71505-250G)
磷酸氢二钠二水合物(Na 2 HPO 4 ·2H 2 O)(Sigma-Aldrich,目录号:71643-250G)
磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P5655-100G)
Trizma碱(Tris(羟甲基)氨基甲烷)(Sigma-Aldrich,目录号:T6791-100G)
85%的正磷酸(H 3 PO 4 )(默克(Merck),目录号:1005731000)
盐酸发烟的37%(HCl)(Merck,目录号:1003171000)
十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L6026-50G)
硼酸(H 3 BO 3 )(Sigma-Aldrich,目录号:B6768-500G)
氢氧化钠颗粒(NaOH)(默克(Merck),目录号:1064821000)
硼氢化钠(NaBH 4 )(Merck,目录号:1063710100)
甲酸(CH 2 O 2 )(默克,目录号:5330020050)
乙腈(CH 3 CN)(Merck,目录号:1000291000)
蛋白酶K 20毫克/毫升(Thermo Scientific的,目录号:EO0491) ,小号撕在-20酶 摄氏
来自球形链霉菌ATCC 21553(Sigma-Aldrich,目录号:SAE0092-10KU)的变溶菌素。重悬于2.5 ml消化缓冲液中(原液浓度〜1 mg / ml),并在-20 °C下储存酶
磷酸盐缓冲盐水(PBS)(请参阅食谱)
裂解缓冲液[ SDS 5%(w / v)] (请参阅食谱)
Tris HCl 1 M pH 8缓冲液(请参见配方)
Tris HCl 100 mM pH 8缓冲液(请参阅食谱)
SDS 10%(w / v)解决方案(请参阅食谱)
消化缓冲液(请参见食谱)
NaOH 2 M溶液(请参阅配方)
硼酸盐缓冲液(请参见食谱)
NaBH 4解决方案(请参阅食谱)
正磷酸25%(V / V)溶液(见ř ecipes)
有机缓冲液A(见ř ecipes)
有机缓冲液B(见ř ecipes)
 
设备
 
移液器(Gilson,目录号s :F144563,F144565,F144566)
玻璃器皿:瓶子,量筒,烧杯
pH计(VWR,目录号:662-1422)
热板搅拌器(VWR,目录号:97042-598)
12 x 6 mm磁力搅拌棒(VWR,货号:442-4501)
微量管离心机(Eppendorf,Centrifuge 5418,目录号:5418000017)
用于15/50 ml锥形管的离心机(Eppendorf,Centrifuge 5920 R,目录号:5948000914)
              贝克曼Optima MAX-TL超速离心机(贝克曼,目录号:A95761)
超速离心机TLA-100.3转子(贝克曼,目录号349490)
真空泵
层流柜
Acquity UPLC系统(Waters)或类似产品(例如,Agilent)
 
软件
 
沃特世Empower 3,版本3471(沃特世,https: //www.waters.com/waters/zh_CN/Empower-3-Chromatography-Data-Software/nav.htm?locale = en_US &cid= 10190669 )
Microsoft Excel版本2004(Microsoft,Office 365,https://www.microsoft.com/zh-cn/microsoft-365/excel? rtc = 1)
Prism 8版本8.0.2(GraphPad,https ://www.graphpad.com/scientific-software/prism/ )
 
程序
 
细胞裂解液的制备
在细菌的合适培养基中将培养物生长到所需的光密度。记录培养物的光密度以进行标准化。为了优化样品制备,目标是10个或10个以上细菌(例如,在1单位OD 600时为10毫升,在0.2单位OD 600时为50毫升)。样品可以按比例放大,但需要更多的试剂,而且更耗时。
为了进行统计分析,请一式三份准备和处理样品。
将培养物转移到50 ml锥形管中,并以3,000 xg收获细胞15分钟。除去上清液,然后将沉淀重悬于1.5 ml自身培养基中。或者,可以将沉淀重悬于PBS或Tris HCl缓冲液中。最终体积并不重要,但最好使用较小的体积,以缩短以后需要的洗涤次数(图1A)。
注意:由于钾盐与SDS反应生成不溶性KDS,因此含钾的重悬介质(例如M63基本介质)可能会干扰针剂的制备。
将样品转移到15 ml锥形管中。这些管必须适合沸腾。在每个管子上放置一个小的搅拌棒(磁铁)(图1A)。
 


图1.肽聚糖样品制备的管线。A.裂解液的制备:收集细菌培养物并将SDS添加到重悬的沉淀中,将其煮沸并搅拌以促进除肽聚糖囊以外的所有细胞组分的溶解。B.葡萄球果用水反复洗涤并进行酶处理。将上清液中释放的多聚肽注射到色谱系统中。C.在UPLC分离过程中应用的流动相梯度。D.通过UPLC梯度分离多肽的步骤。
 
加入1.5 ml裂解缓冲液,然后将试管放在装有烧水的烧杯中,并置于磁性热搅拌板上。将样品煮沸30分钟至2小时,然后关闭加热板,让裂解液搅拌过夜。细胞在SDS中煮沸后会立即裂解,但是建议使用更长的搅拌时间来剪切DNA (图1A)。
煮沸的裂解物可以在室温下长时间保存。确保盖紧,避免干燥。
 
隔离和正革兰氏阴性菌肽聚糖
要完全去除SDS,请通过离心水进行几次洗涤。使用3 ml聚碳酸酯超速离心管将样品旋转10 min和150,000 xg 。由于SDS会在低温下沉淀,因此请使用20 °C或更高的温度。用3 ml裂解液填充试管。确保转子正确平衡。离心后,所有可溶性化合物将保留在上清液中。所得的颗粒通常是透明的并且难以区分。建议在试管的一侧做标记,并以此作为定位沉淀的参考(图1B)。
注意:如果在清洗过程中观察到白色沉淀(可能不溶的KDS),请使用Tris HCl 100 mM pH 8.0缓冲液代替水。
用真空泵小心丢弃上清液,而不会除去沉淀物。将沉淀重悬于900 µl MilliQ水中,并通过评估气泡的产生来检查SDS的存在。如果需要,加入2 ml MilliQ水,混合并再次离心。重复此洗涤步骤,直到SDS被完全清除(将沉淀重悬于水中,通常2-4次洗涤后,不会形成气泡)。
要去除Braun脂蛋白或其他与PG相关的蛋白,请用蛋白酶处理冲洗后的囊肿。此步骤是可选的,但强烈建议这样做。将重悬的沉淀(〜900 µl)转移到2 ml的微管中。加入100 µl Tris HCl 1 M pH 8缓冲液和1 µl蛋白酶K 20 mg / ml。在37 °C下孵育样品1小时。加入110 µl 10%(w / v)的SDS终止反应,并在水中煮沸5分钟。
注意:蛋白酶K是一种广谱丝氨酸蛋白酶,用于去除PG结合蛋白(例如布劳恩脂蛋白)。该酶在1%(w / v)的SDS中保留活性。一些细菌不需要蛋白酶K处理,但是建议去除其他与PG相关的蛋白。也可以使用其他蛋白酶,如胰凝乳蛋白酶(EC 3.4.21.1)或胰蛋白酶(EC 3.4.21.4)。             
让样品冷却,然后再次转移到超速离心管中。加入2 ml MilliQ水洗涤蛋白酶K消化的囊皮。如前所述混合并超速离心。小心丢弃上清液,并在完全除去SDS后,将沉淀重悬于50-100 µl消化缓冲液中。最终体积至关重要,因为在muramidase消化后可溶性多聚肽将保留在可溶性级分中。洗涤后的葡萄球菌应保存在4 °C以避免降解或污染。不建议将样品保存在-20 °C,因为由于冷冻/融化过程,气泡可能会破裂。
 
村酰胺酶消化
将重悬液(50-100 µl)转移至1.5 ml微管中。
              加入2 µl 1 mg / ml mutanolysin,使反应在37 °C下工作2-16 h 。2小时后反应几乎完成,但要确保完全消化,请使其进行更长的时间。
要使酶失活,请b将样品上油5分钟。请勿在样品中添加任何清洁剂。
在室温下离心15分钟,在台式离心机中离心20,000 xg ,并将上清液转移到新的1.5 ml微管中。该上清液包含由变溶菌素释放的可溶性多聚肽(图1B)。这些样品非常稳定,可以在4或-20 °C下保存。
 
减少样品
注意:减少样品量是可选的,但强烈建议使用。用变溶素消化可产生终末还原NAM。样品的减少避免了不同的端基构型,并避免了在PG谱中每个muropeptide出现2至4个峰,因为NAM被还原为muramitol。在层流机柜中执行此过程。如果未进行还原,则用稀释的正磷酸(0.25%,v / v)将样品的pH调节至2.0-4.0 。
将硼酸盐缓冲液添加到样品中以将pH值调节到8.5-9.0。对于100 µl反应,通常使用15-20 µl硼酸盐缓冲液。使用指示条检查pH:取0.5 µl样品,滴在指示条上。检查参考表上的颜色/ pH。在高pH不要离开样品太久,因为它会导致碱性在剔除(自卸车,1968年)。
加入20 µl新鲜制备的NaBH 4 2 M,并在室温下将样品还原30分钟。该反应产生H 2并积聚气泡,请勿关闭管盖以防止气体积聚。
用25%的正磷酸(v / v)将样品的pH调节至2.0-4.0。添加酸会引起剧烈反应并突然形成气泡。首先添加4 µl,并按照前面所述用适当的指示条检查pH值。如果需要,小心地以微升加入微升酸,以确保样品达到所需的pH。
以最大速度旋转试管1分钟,以确保最大的样品回收率。将样品转移到96孔V形底部微孔板中。使用适当的粘合剂密封垫密封板。样品可以在-20 °C下长期保存。可能会形成不溶性沉淀物,如果观察到,则应在进样到UPLC中之前过滤样品。
 
UPLC分离
注意:设置取决于UPLC的软件,型号和制造商。对于多肽谱分析,反相(RP)色谱柱,通常能够保留和分离中极性和非极性代谢物的C18键合硅胶提供了良好的分离模式。对于RPLC,通过使用低电溶强度(即主要或完全由水组成)的溶剂将样品上样到色谱柱上,可以确保最大程度地保留分析物。保留的代谢物的洗脱是通过增加乙腈含量的梯度来完成的。
将色谱柱温度设置为45°C。
与有机缓冲液A制备流动相和再填充的瓶子A和B [甲酸0.1%(V / V)]和有机缓冲液B [乙腈甲酸0.1%(V / V)]分别。根据UPLC系统说明清洗泵和管道。
用有机缓冲液A平衡色谱柱,流速0.25 ml / min,直到压力稳定。
使用系统自动进样器,进样10 µl样品。
执行使用图中描述的梯度的LC运行小号1C和1 d在204纳米和测量吸光度。
运行空白进样以监控并减去基线。
 
故障排除
严格按照制造商的指示控制参数,例如流速和柱温,以确保分离的可重复性。典型地,〜2000个样品可以在色谱柱中注入之前分离效率丢失,峰小号降解和保留时间小号变化。如果色谱图显示出不良的峰形,则表明色谱柱降解或样品超载。要解决此问题,请稀释样品或改进样品制备。如果问题仍然存在,请考虑清洁或更换色谱柱。             
              可以根据样品浓度修改进样量:
对于浓缩样品(例如,来自大型起始培养物的PG),应注入较少的体积,以确保系统在其检测限内运行。
如果没有或只有很少的峰或信号太低,请增加进样量,使用Speedvac浓缩器浓缩样品,或通过在变溶酶消化之前将洗涤后的囊泡以较小的体积重悬来制备新样品。
保留时间的变化可能是由于样品pH值不合适或样品中存在清洁剂(SDS)所致。当样品的pH值大于5时,色谱图会移动。用25%(v / v)的正磷酸调节pH值,然后重新运行样品。如果样品中有清洁剂,则准备新的样品以增加洗涤步骤,或在每次运行后洗涤色谱柱以除去残留的清洁剂。
不适当的样品减少会导致保留时间发生变化。重复样品制备,添加过量的NaBH 4 。
在运行之间被其他组分污染或重新平衡时间短也会导致出现鬼峰。
              当样品浓度低时,基线漂移和噪声变得更加明显。用水进行空白进样,并在数据处理期间减去基线。
 
数据分析
 
一式三份准备并运行样品以进行统计分析。
为了获得最佳的比较条件,请对所有样品使用相同的体积,洗涤量和处理量。
 
肽聚糖谱
提取原始数据:保留时间(分钟)和204 nm处的吸光度(任意单位)。
对于基线校正,减去空白水注入的数据(图2A)。
定义色谱处理区域,删除无用的数据,通常是进样前和运行结束时的清洗(数据点≤2-3分钟,数据点≥12-13分钟)(图2A)。
通过将204 nm(任意单位)处的吸光度相对于保留时间(分钟)作图来表示色谱图(图2A)。
 


图2.肽聚糖数据分析示意图。A.转换原始数据以获得最佳吸光度与时间的关系。减去空白进样量以进行基线校正,并修剪要显示的数据点。B.简单的肽聚糖色谱图的例子。C.用于定量多肽相对摩尔丰度和交联的分子式。D.表显示了示例概况中的多肽丰度。E.从多肽丰度表中计算得出的肽聚糖特征。
 
通过与在相同色谱系统中运行的已知参考样品进行比较来鉴定多肽。未知或新颖的峰只能通过质谱分析来鉴定。
 
相对总PG量
为了进行归一化,通过将收集时记录的光密度除以对照样品的平均光密度来计算相对细胞数量。
通过将感兴趣的时间范围(最小2-3到最小12-13)中的所有强度值相加,计算色谱图的总面积。
通过将每个样本的总面积除以其相对细胞数量来归一化总面积。
将标准化总面积除以对照样品的平均值,然后将结果乘以100,以计算相对百分比。
执行未配对的t检验分析,以确定结果的统计显着性。
 
巯肽定量
为了进行定量,请使用适当的软件(例如UPLC制造商软件,MATLAB)通过积分计算每个峰的面积(图2B)。
为了计算峰摩尔丰度,将每个面积除以多肽摩尔比:为简单起见,如果是单体,则为1;如果是二聚体,则为2;如果是三聚体,则为3;……(图2C)。
确定每个峰的相对摩尔丰度:将每个峰的摩尔丰度除以色谱图的总和(所有单个摩尔丰度的总和),然后将结果乘以100以计算相对百分比(图2C)。
执行未配对的t检验分析,以确定结果的统计显着性。
用多肽表表示结果,该表通常包含所有检测到的多肽的保留时间和相对摩尔丰度(图2D)。
 
肽聚糖主要特征的计算
单体(%):所有单体多聚肽的相对摩尔丰度之和。
二聚体(%):所有二聚体巨肽的相对摩尔丰度之和。
三聚体(%):所有三聚体巨肽的相对摩尔丰度之和。
交联(%):计算为Dimers + (Trimers × 2)的交联百分比。二聚体中有交联,三聚体中有2个交联(图2C)。
脱水(%):将所有脱水muropepteptes(具有(1-6anhydro)MurNAc的mueptepteptides)的相对摩尔丰度相加。
链长(聚糖单位):通过将100除以脱水多肽的百分比来计算平均聚糖链长。
执行未配对的t检验分析,以确定结果的统计显着性。
将结果表示为PG功能表(图2D)。
 
PG分析的真实示例如图3所示。
 


图3.具有不同阻抑水平的PBP1b和/或PBP1a的大肠杆菌样品的肽聚糖分析。A.代表性的UPLC配置文件。指出了鉴定的多肽。B.相对PG量(与AV84 5-5比较)。C.交联的百分比。计算公式为:二聚体+(三聚体× 2)。D.主要的PG特征显示单体,二聚体和三聚体的相对摩尔丰度,总交联值,脱水多肽的相对摩尔量和估计的链长。统计分析:不成对的t检验。NS:不重要。数据代表平均值 ± SEM,3个副本。数据来自Vigouroux等修改。,2020年。
 
笔记
 
不建议使用此方案从mos g革兰氏阳性细菌制备PG ,因为存在相关的细胞壁聚合物(例如壁板壁酸或脂壁壁酸)需要更精细的去除程序(Mann等人,2016) 。
该协议也适用于裂解的样品(例如,用抗生素,例如D-环丝氨酸处理),只要在收集样品以制备球囊之前进行了超速离心步骤:
在4 °C和200,000 xg下旋转样品30分钟,并丢弃上清液。
将对应于不溶部分的沉淀重悬于1.5 ml上清液或其他培养基中。
继续执行步骤A3。
 
菜谱
 
用新鲜的MilliQ水准备所有解决方案。在25 °C下,使用来自蒸馏或去离子装置的超纯水,电阻为8MΩ/ cm 。对于UPLC缓冲液,仅使用分析纯试剂。由于pH在大多数所用溶液中至关重要,因此请确保轻轻搅拌溶液并在与工作条件尽可能相近的温度下调节pH。溶液在室温下保存。
 
磷酸盐缓冲液(PBS)
将8 g NaCl,0.2 g KCl,1.77 g Na 2 HPO 4 ·2H 2 O和0.24 g KH 2 PO 4溶解在800 ml MilliQ水中
搅拌直至完全溶解
用HCl调节至pH 7.4
用MilliQ水将最终体积调节至1 L
高压灭菌
裂解缓冲液[ SDS 5%(w / v)]
将5 g SDS溶于100 ml MilliQ水中
注意:使用防护装备是因为SDS粉末可能会刺激呼吸道。
              Tris HCl 1 M pH 8.0缓冲液
将12.11 g Tris碱溶于80 ml MilliQ水中
搅拌直至完全溶解
用HCl调节至pH 8.0
用MilliQ水将最终体积调节至100 ml
              Tris HCl 100 mM pH 8.0缓冲液
在80 ml MilliQ水中稀释10 ml Tris HCl 1 M pH 8缓冲液             
用MilliQ水将最终体积调节至100 ml
SDS 10%(w / v)解决方案
将10 g SDS溶于100 ml MilliQ水中
注意:使用防护装备是因为SDS粉末可能会刺激呼吸道。
消化缓冲液(50 mM磷酸盐缓冲液,pH 4.9)
将887.6 mg NaH 2 PO4·2H 2 O和9.5 mg Na 2 HPO 4 ·2H 2 O溶于80 ml MilliQ水中
搅拌直至完全溶解
用正磷酸25%(v / v)溶液将pH调节至4.9以精细调节pH
用MilliQ水将体积调节至100 ml
NaOH 2 M溶液
将8 g NaOH溶于100 ml MilliQ水中,搅拌直至完全溶解
笔记:
该反应是高放热反应,必须采取安全措施。
NaOH溶液相对不稳定,其浓度会随时间变化,因此为了获得更高的精确度,最好准备新鲜的溶液。
硼酸盐缓冲液(硼酸盐缓冲液0.5 M pH 9.0)
将3.1克硼酸溶于80毫升MilliQ水中,搅拌直至完全溶解
用NaOH 2 M溶液将pH调节至9.0
用MilliQ水将最终体积调节至100 ml
NaBH 4溶液(NaBH 4 2 M)
将76 mg NaBH 4溶于1 ml MilliQ水中
注意:请勿关闭任何管盖,因为该还原剂会与溶剂发生剧烈反应而生成H 2 。此解决方案必须新鲜准备,请勿存放。
正磷酸25%(v / v)溶液
在50 ml MilliQ水中稀释29.4 ml正磷酸85%
搅拌直至完全稀释
用MilliQ水将最终体积调节至100 ml
有机缓冲液A [甲酸0.1%(v / v)]
在1升MilliQ水中稀释1毫升HPLC级甲酸并混合
有机缓冲液B [乙腈中的甲酸0.1%(v / v)]
将1毫升HPLC级甲酸在1升乙腈中稀释并混合
 
致谢
 
这项工作详细报告了革兰氏阴性细菌的肽聚糖样品的样品制备和分析。这项工作得到了Knut和Alice Wallenberg基金会,瑞典研究委员会,Kempe基金会以及瑞典分子感染医学实验室的支持。Stiftelsen Clas Groschinskys Minnesfond的项目资助也得到了认可。巴斯德研究所的工作得到了欧洲研究理事会(ERC)的支持,该研究属于欧盟的Horizon 2020研究与创新计划[授权协议号(679980)],法国政府的Avenir研究计划,卓越的“综合生物学”实验室新兴传染病”(ANR-10-LABX-62-IBEID),巴黎圣母院“新兴”计划和大众基金会的资助。资助者在研究设计,数据收集和解释或将作品提交发表的决定中没有作用。该方案源自我们工作中发表的大肠杆菌PG分析方法(Vigouroux等,2020)。
 
利益争夺
 
作者声明没有利益冲突或竞争利益。
 
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Copyright Alvarez et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Alvarez, L., Cordier, B., van Teeffelen, S. and Cava, F. (2020). Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography. Bio-protocol 10(19): e3780. DOI: 10.21769/BioProtoc.3780.
  2. Vigouroux, A., Cordier, B., Aristov, A., Alvarez, L., Ozbaykal, G., Chaze, T., Oldewurtel, E. R., Matondo, M., Cava, F., Bikard, D. and van Teeffelen, S. (2020). Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects. Elife 9: e51998.
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