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Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS

HPLC-MS分析N-乙酰胞壁酸-6-磷酸（MurNAc-6P）的积累

MB Marina Borisova
CM Christoph Mayer

发布: 2017年08月05日第7卷第15期 DOI: 10.21769/BioProtoc.2420 浏览次数: 8691

评审: Masahiro MoritaKanika GeraAnonymous reviewer(s)

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Abstract

We describe here in detail a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based method to determine N-acetylmuramic acid-6-phosphate (MurNAc-6P) in bacterial cell extracts. The method can be applied to both Gram-negative and Gram-positive bacteria, and as an example we use Escherichia coli cells in this study. Wild type and mutant cells are grown for a defined time in a medium of choice and harvested by centrifugation. Then the cells are disintegrated and soluble cell extracts are generated. After removal of proteins by precipitation with acetone, the extracts are analyzed by HPLC-MS. Base peak chromatograms of wild type and mutant cell extracts are used to determine a differential ion spectrum that reveals differences in the MurNAc-6P content of the two samples. Determination of peak areas of extracted chromatograms of MurNAc-6P ((M-H)- = 372.070 m/z in negative ion mode) allows quantifying MurNAc-6P levels, that are used to calculate recycling rates of the MurNAc-content of peptidoglycan.

Keywords: Bacteria (细菌) search Cell wall metabolism (细胞壁代谢) search Peptidoglycan recycling (肽聚糖再循环) search Cytosolic metabolites (胞质代谢物) search LC-MS (LC-MS) search Base peak chromatogram (BPC) (基峰色谱图（BPC）) search Extracted ion chromatogram (EIC) (萃取离子色谱图（EIC）) search MurNAc-6P accumulation (MurNAc-6P累积) search

Background

Large parts of the peptidoglycan cell wall of bacteria are steadily turned over and possibly recovered (recycled) during bacterial growth. A key compound of the peptidoglycan recycling metabolism is N-acetylmuramic acid-6-phosphate (MurNAc-6P), which accumulates in a MurNAc-6P etherase (MurQ) mutant of Escherichia coli (Jaeger et al., 2005; Uehara et al., 2006). MurQ orthologs are found in many bacteria, including Gram-positive bacteria (Litzinger et al., 2010; Reith and Mayer, 2011). MurNAc-6P accumulation in murQ mutants recently proved recycling of the MurNAc-content of the bacterial cell wall in Gram-positive bacteria and was used to quantify intracellular MurNAc-6P levels, which allowed determining peptidoglycan recycling rates (Borisova et al., 2016).

Materials and Reagents

  1. 50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
  2. Micro-tubes 2 ml (SARSTEDT, catalog number: 72.691 )
  3. Micro-tubes 2 ml with cap (SARSTEDT, catalog number: 72.694 )
  4. Glass beads (0.25 to 0.5 mm) (Carl Roth, catalog number: A553.1 )
  5. Escherichia coli strains: MC4100 (wild type) and TJ2e (∆murQ) (Jaeger et al., 2005)
  6. Acetone (CH3COCH3) (Sigma-Aldrich, catalog number: 34850-2.5L )
  7. Ammonium formate (NH4HCOO) (VWR, catalog number: 17843-50G )
  8. Acetonitrile (CH3CN) (Avantor Performance Materials, J.T. Baker®, catalog number: 9012-03 )
  9. Millipore ultrapure water (autoclaved)
  10. BactoTM yeast extract (BD, BactoTM, catalog number: 212720 )
  11. BactoTM tryptone (BD, BactoTM, catalog number: 211699 )
  12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-5KG-R )
  13. Propan-2-ol (Sigma-Aldrich, catalog number: 34863-2.5L-M )
  14. Formic acid (HCOOH) (VWR, catalog number: 56302-50ML )
  15. Sodium hydroxide (NaOH), 1 N (VWR, catalog number: 31627.290 )
  16. Medium Luria Bertani (LB) broth (see Recipes)
  17. LC-MS calibrant (10 mM sodium formate) (see Recipes)
  18. HPLC buffer A (see Recipes)

Equipment

  1. 1,000 ml Erlenmeyer flasks with chicane
  2. 100 ml Erlenmeyer flasks with chicane
  3. Shaker (set at 160 rpm) (Eppendorf, New BrunswichTM, model: Excella® E10 )
  4. Pipette controller (BrandTech Scientific, model: accu-jet® pro )
  5. Microcentrifuge (Thermo Fischer Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 )
  6. Medium bench centrifuge (Thermo Fischer Scientific, model: HeraeusTM Biofuge Pico )
  7. Cell density meter (Biochrom, model: Biochrom WPA CO8000 )
  8. Cell disrupter (GMI, model: Thermo Savant FastPrep 120 )
  9. Rotational vacuum concentrator (Martin Christ Gefriertrocknungsanlagen, model: RVC 2-18 CDplus )
  10. Gemini® 5 µm 110Å, 150 x 4.6 mm LC column (Phenomenex, catalog number: 00F-4435-E0 )
  11. Mass spectrometer (Bruker, model: micrOTOF focus II )
  12. High-performance liquid chromatography (Thermo Fischer Scientific, Thermo ScientificTM, model: UltimateTM 3000 RS )

Software

  1. Chromeleon Xpress (Dionex)
  2. MicroTOF control Version 3.0 (Bruker Daltonics)
  3. Bruker Compass HyStar Version 3.2 (Bruker Daltonics)
  4. Compass Data Analysis Version 4.0 (Bruker Daltonics)
  5. MetaboliteDetect 2.0 (Bruker Daltonics)
  6. GraphPad Prism 6 (San Diego, CA, USA)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Borisova, M. and Mayer, C. (2017). Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS. Bio-protocol 7(15): e2420. DOI: 10.21769/BioProtoc.2420.

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分类

微生物学 > 微生物生理学 > 膜性能

微生物学 > 微生物生物化学 > 其它化合物

生物化学 > 糖类 > 肽聚糖

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