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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   

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

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

  1. Bacterial growth
    1. Add 20 ml LB medium to a 100 ml Erlenmeyer flask with chicane.
    2. Inoculate LB broth media with single colonies of E. coli wild type and ∆murQ grown on agar plates, which were streaked out the day before.
    3. Grow bacteria for 16 h at 37 °C and 160 rpm.
    4. Measure OD600 nm of the overnight cultures (expected OD of 3.5 for wild type and ∆murQ).
    5. Add 200 ml LB medium to 1,000 ml Erlenmeyer flasks.
    6. Inoculate the 200 ml LB medium with bacteria to obtain an initial OD600 nm of 0.05.
    7. After 3 h of growth, bacteria are expected to reach an OD600 nm of 1.76.

  2. Generation of bacterial cytosolic fractions
    1. Spin down 170 ml (a volume corresponding to 100 ml OD 3) of bacterial suspension (4 x 50 ml Falcons) at 3,000 x g for 10 min at room temperature.
    2. Carefully resuspend bacterial pellets in 20 ml Millipore water. Avoid vortexing of the bacterial suspension to prevent cell lysis.
    3. Spin down bacteria at 3,000 x g for 10 min at room temperature.
    4. Discard supernatant and freeze pellets immediately at -80 °C. Samples are further proceeded the next day or within one week of storage at -80 °C.
    5. Thaw frozen samples at room temperature and resuspend pellets in Millipore water to a final volume of 1.2 ml.
    6. Add 0.25 g of the glass beads to the Micro-tubes with cups.
    7. Transfer 1.2 ml bacterial suspension to the micro tubes with glass beads.
    8. Disintegrate cells with glass beads using a cell disruptor (4x for 35 sec at speed 6). After the second cycle, chill cells down for 1 min on ice.
    9. Spin down samples in a microcentrifuge at 16,000 x g for 10 min at room temperature.
    10. Add 200 µl of the supernatant to 800 µl of ice-cold acetone to precipitate remaining proteins in the samples in 2 ml Micro-tubes and invert tubes 3 times.
    11. Centrifuge samples at 16,000 x g for 10 min at room temperature and transfer supernatant to a new 2 ml Micro-tube.
    12. Dry cytosolic fractions under vacuum for 2 h at 55 °C and store at 4 °C.
    13. Dissolve cytosolic fractions in 100 µl Millipore water prior to LC-MS measurements.
    14. Inject 5 µl of each sample to the LC column, pre-equilibrated with buffer A (0.1% formic acid, 0.05% ammonium formate).

  3. LC-MS program
    1. Generate with MicrOTOF control an MS program applying a mass range of 80 to 3,000 m/z.
    2. Calibrate MS in negative ion mode using 10 mM sodium formate calibrant.
    3. Generate with Bruker Compass HyStar a 45-min-HPLC gradient program:
      1. Flow rate of 0.2 ml/min.
      2. Column compartment temperature 37 °C.
      3. UV trace of 202 nm.
      4. 5 min 100% buffer A (0.1% formic acid, 0.05% ammonium formate).
      5. 30 min linear gradient from 100% to 60% buffer A (40% buffer B [100% acetonitrile]).
      6. 5 min 60% buffer A.
      7. 5 min 100% buffer A.

Data analysis

HPLC-MS data for E. coli wild type (WT) and ∆murQ mutant samples were analyzed with Compass Data Analysis. Data are shown as base peak chromatograms (BPC) in negative ion mode within a mass to charge (m/z) range of 80 to 3,000. A differential ion spectrum (DS) was generated, subtracting the BPC of wild type from the BPC of ∆murQ (Figure 1), using the program Metabolite Detect and a difference factor of 5. The DS revealed major differences in intracellular metabolite levels at a retention time of 19.8 to 22.7 min (Figure 1). The DS at this retention time contains ions in negative ion mode corresponding to MurNAc-6P ((M-H)- = 372.071 m/z), an elimination product of MurNAc-6P (282.039 m/z), and MurNAc-6P dimer (745.148 m/z) (Figure 2). Furthermore, extracted ion chromatograms (EICs) for MurNAc-6P ((M-H)- calculated = 372.070 m/z) were generated using the Compass Data Analysis tool. The area under the curve (AUC) for the EIC of MurNAc-6P was determined using the program Prism 6 with the baseline set to 30 (Figure 3). Analysis of the AUC for the EICs for MurNAc-6P in combination with a standard curve can be used to quantify the amount of MurNAc-6P accumulating in ∆murQ mutants (Borisova et al., 2016).


Figure 1. HPLC-MS analyses of soluble extracts of WT and ∆murQ mutant cells. Base peak chromatograms (BPC) show similar metabolite pattern, with differences that can be visualized by calculating a differential ion spectrum (DS).


Figure 2. Mass spectrum of the differential ion spectrum (DS) signal at 19.8 to 22.7 min of Figure 1. The compound is identified as MurNAc-6P by its exact mass ((M-H)- observed = 372.071 m/z, theoretical = 372.070 m/z), the exact mass of a dimer (745.148 m/z) and of an elimination product (282.039 m/z).


Figure 3. Extracted ion chromatogram (EIC) for MurNAc-6P. Searching for an EIC of 372.071 m/z of MurNAc-6P revealed no signal for wild type (WT) cells (interrupted black line) but a clear signal for ∆murQ mutant cells (blue line; upper panel). The area under the curve (AUC) for the latter signal allows to quantify the amount of MurNAc-6P using a MurNAc-6P standard of known concentration (lower panel) (Unsleber et al., 2017).

Notes

  1. Use only HPLC grade chemicals and ultrapure Millipore water for sample preparation and mass spectrometry analysis.
  2. Wash bacterial cultures extensively with Millipore water to remove contamination of salt and components from the LB broth medium.
  3. Avoid vortexing of the bacterial suspension during washing to prevent cell lysis.
  4. Freeze bacterial cultures at -80 °C to improve subsequent cell disruption with the glass beads.
  5. Use ice-cold acetone to precipitate efficiently remaining proteins in the cytosolic fractions prior to LC-MS measurements.
  6. Perform detection of MurNAc-6P in negative ion mode. MurNAc-6P could not be detected in positive ion mode.

Recipes

  1. Medium Luria-Bertani (LB) broth, Miller
    5 g yeast extract
    10 g tryptone
    10 g sodium chloride
    in 1 L distilled water
    Autoclave medium at 121 °C for 15 min
  2. LC-MS calibrant (10 mM sodium formate)
    12.5 ml Millipore water
    12.5 ml propan-2-ol
    50 µl formic acid
    250 µl 1 N NaOH
  3. HPLC buffer A: 0.1% formic acid, 0.05% ammonium formate (pH 3.2)
    1 ml formic acid
    0.5 g ammonium formate
    1 L autoclaved Millipore water

Acknowledgments

This protocol was adapted from Borisova et al. (2016). Christoph Mayer is supported by the Deutsche Forschungsgemeinschaft (DFG) grants MA2436/7, SFB766/A15 and GRK1708/B2.

References

  1. Borisova, M., Gaupp, R., Duckworth, A., Schneider, A., Dalugge, D., Mühleck, M., Deubel, D., Unsleber, S., Yu, W., Muth, G., Bischoff, M., Götz, F. and Mayer, C. (2016). Peptidoglycan recycling in Gram-positive bacteria is crucial for survival in stationary phase. MBio 7(5).
  2. Jaeger, T., Arsic, M. and Mayer, C. (2005). Scission of the lactyl ether bond of N-acetylmuramic acid by Escherichia coli "etherase". J Biol Chem 280(34): 30100-30106.
  3. Litzinger, S., Duckworth, A., Nitzsche, K., Risinger, C., Wittmann, V. and Mayer, C. (2010). Muropeptide rescue in Bacillus subtilis involves sequential hydrolysis by β-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine amidase. J Bacteriol 192(12): 3132-3143.
  4. Reith, J. and Mayer, C. (2011). Peptidoglycan turnover and recycling in Gram-positive bacteria. Appl Microbiol Biotechnol 92(1): 1-11.
  5. Uehara, T., Suefuji, K., Jaeger, T., Mayer, C. and Park, J. T. (2006). MurQ Etherase is required by Escherichia coli in order to metabolize anhydro-N-acetylmuramic acid obtained either from the environment or from its own cell wall. J Bacteriol 188(4): 1660-1662.
  6. Unsleber, S., Borisova, M. and Mayer, C. (2017). Enzymatic synthesis and semi-preparative isolation of N-acetylmuramic acid 6-phosphate. Carbohydr Res 445: 98-103.

简介

我们在这里详细描述了一种基于高效液相色谱 - 质谱(HPLC-MS)的方法,以确定细菌细胞提取物中的N-乙酰谷氨酸-6-磷酸(MurNAc-6P)。该方法可以应用于革兰氏阴性菌和革兰氏阳性菌,作为一个例子,我们在本研究中使用大肠杆菌细胞。野生型和突变型细胞在选择的培养基中生长一定时间并通过离心收获。然后细胞分解并产生可溶性细胞提取物。通过用丙酮沉淀除去蛋白质后,通过HPLC-MS分析提取物。野生型和突变型细胞提取物的基本峰图谱用于测定显示两种样品中MurNAc-6P含量差异的差异离子谱。确定阴离子模式下MurNAc-6P((MH)= 372.070m/z的提取色谱图的峰面积允许定量使用的MurNAc-6P水平计算消泡聚糖的MurNAc含量的回收率。
【背景】在细菌生长期间,大部分细菌的肽聚糖细胞壁稳定地翻转并可能回收(再循环)。 肽聚糖回收代谢的关键化合物是积聚在大肠杆菌的MurNAc-6P醚酶(MurQ)突变体中的N-乙酰谷氨酸-6-磷酸(MurNAc-6P) (Jaeger等人,2005; Uehara等人,2006)。 MurQ直向同源物被发现在许多细菌中,包括革兰氏阳性菌(Litzinger等人,2010; Reith和Mayer,2011)。 MurNAc-6P突变体中的MurNAc-6P积累最近证明在革兰氏阳性细菌中循环使用细菌细胞壁的MurNAc含量,并用于定量细胞内MurNAc-6P水平,这允许测定肽聚糖回收率( Borisova 等,2016)。

关键字:细菌, 细胞壁代谢, 肽聚糖再循环, 胞质代谢物, LC-MS, 基峰色谱图(BPC), 萃取离子色谱图(EIC), MurNAc-6P累积

材料和试剂

  1. 50ml管(SARSTEDT,目录号:62.547.254)
  2. 微管2 ml(SARSTEDT,目录号:72.691)
  3. 微管2毫升带帽(SARSTEDT,目录号:72.694)
  4. 玻璃珠(0.25至0.5毫米)(Carl Roth,目录号:A553.1)
  5. 大肠杆菌菌株:MC4100(野生型)和TJ2e(Δκα),(Jaeger等人,2005)
  6. 丙酮(CH 3 COCH 3)(Sigma-Aldrich,目录号:34850-2.5L)
  7. 甲酸铵(NH 4 HCOO)(VWR,目录号:17843-50G)
  8. 乙腈(CH 3 N CN)(Avantor Performance Materials,J.T.Baker,目录号:9012-03)
  9. Millipore超纯水(高压灭菌)
  10. Bacto TM 酵母提取物(BD,Bacto TM,目录号:212720)
  11. Bacto TM 胰蛋白胨(BD,Bacto TM ,目录号:211699)
  12. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434-5KG-R)
  13. 丙-2-醇(Sigma-Aldrich,目录号:34863-2.5L-M)
  14. 甲酸(HCOOH)(VWR,目录号:56302-50ML)
  15. 氢氧化钠(NaOH),1N(VWR,目录号:31627.290)
  16. 中等Luria Bertani(LB)肉汤(见食谱)
  17. LC-MS校准物(10mM甲酸钠)(参见食谱)
  18. HPLC缓冲液A(参见食谱)

设备

  1. 1000毫升锥形瓶与chicane
  2. 100毫升锥形瓶与chicane
  3. 振荡器(设定在160rpm)(Eppendorf,New Brunswich TM ,型号:Excella ® E10)
  4. 移液器控制器(BrandTech Scientifc,型号:accu-jet ® pro)
  5. 微量离心机(Thermo Fischer Scientific,Thermo Scientific TM ,型号:Heraeus TM Pico TM 17)
  6. 中型台式离心机(Thermo Fischer Scientific,型号:Heraeus TM Biofuge Pico)
  7. 细胞密度计(Biochrom,型号:Biochrom WPA CO8000)
  8. 细胞破坏器(GMI,型号:Thermo Savant FastPrep 120)
  9. 旋转式真空浓缩机(Martin Christ Gefriertrocknungsanlagen,型号:RVC 2-18 CDplus)
  10. Gemini ® 5μm110Å,150 x 4.6 mm LC色谱柱(Phenomenex,目录号:00F-4435-E0)
  11. 质谱仪(Bruker,型号:micrOTOF焦点二)
  12. 高性能液相色谱(Thermo Fischer Scientific,Thermo Scientific TM,型号:Ultimate TM 3000 / RS)

软件

  1. Chromeleon Xpress(Dionex)
  2. MicroTOF控制版3.0(Bruker Daltonics)
  3. Bruker Compass HyStar版本3.2(Bruker Daltonics)
  4. 指南针数据分析版本4.0(Bruker Daltonics)
  5. MetaboliteDetect 2.0(Bruker Daltonics)
  6. GraphPad Prism 6(San Diego,CA,USA)

程序

  1. 细菌生长
    1. 将20ml LB培养基加入到具有chicane的100ml锥形瓶中
    2. 接种具有单个殖民地的LB肉汤培养基。大肠杆菌野生型和Δ琼脂生长在琼脂平板上,其前一天被划痕。
    3. 在37℃和160rpm下生长细菌16小时。
    4. 测量过夜培养物的OD 600nm(野生型的预期OD为3.5和MurQ)。
    5. 加入200毫升LB培养基至1,000毫升锥形瓶
    6. 接种200ml含有细菌的LB培养基以获得0.05的初始OD 600nm。
    7. 生长3小时后,预计细菌达到1.76的OD 600nm
  2. 产生细菌胞质级分
    1. 在室温下将3,000ml(相当于100ml OD 3的体积)的细菌悬浮液(4×50ml Falcons)以3,000×g旋转10分钟。
    2. 小心地将细菌沉淀在20ml Millipore水中。避免涡旋的细菌悬浮液,以防止细胞裂解。
    3. 在室温下以3,000×g将细菌分解10分钟
    4. 弃去上清液,并立即在-80℃冷冻颗粒。样品在第二天或-80°C储存后一周内进一步进行。
    5. 在室温下将冷冻样品解冻,并将Millipore水中的沉淀重新悬浮至最终体积为1.2ml
    6. 用杯子将0.25g玻璃珠加入到微管中
    7. 将1.2ml细菌悬浮液转移到带玻璃珠的微管上
    8. 使用细胞破坏剂将细胞与玻璃珠分解(4x,速度为35秒,速度为6)。第二个循环后,在冰上冷却细胞1分钟
    9. 将微量离心机中的样品以16,000 x g在室温下旋转10分钟。
    10. 将200μl上清液加入800μl冰冷的丙酮中,将样品中的剩余蛋白质沉淀在2 ml微管中,倒置3次。
    11. 在室温下以16,000×g离心样品10分钟,并将上清液转移到新的2ml微管中。
    12. 在55℃真空干燥细胞质级分2小时并储存在4℃。
    13. 在LC-MS测量之前,将细胞溶质级分溶解于100μlMillipore水中
    14. 将5μl每个样品注入LC柱,预先用缓冲液A(0.1%甲酸,0.05%甲酸铵)平衡。

  3. LC-MS程序
    1. 使用MicrOTOF生成控制MS程序,应用质量范围为80到3,000 m / z 。
    2. 使用10mM甲酸钠校准物校正MS在负离子模式
    3. 用Bruker Compass HyStar生成45分钟HPLC梯度程序:
      1. 流速为0.2 ml / min
      2. 柱室温度37°C
      3. 202nm的紫外线踪迹
      4. 5分钟100%缓冲液A(0.1%甲酸,0.05%甲酸铵)
      5. 30%线性梯度从100%至60%缓冲液A(40%缓冲液B [100%乙腈])
      6. 5分钟60%缓冲区A.
      7. 5分钟100%缓冲液A.

数据分析

E-E的HPLC-MS数据。使用指南针数据分析分析大肠杆菌野生型(WT)和ΔhIQQ突变体样品。数据显示为负离子模式下的基峰峰值色谱图(BPC),其质量要达到80至3000的质量(m / z / em)范围。产生差示离子谱(DS),使用程序代谢检测法和差异因子5从BPC中减去野生型BPC(图1),差异因子为5.DS显示主要在保留时间为19.8至22.7分钟时细胞内代谢物水平的差异(图1)。在该保留时间的DS包含对应于MurNAc-6P((MH))的负离子模式的离子,MurNAc-6P的消除产物(282.039m / z)和MurNAc-6P二聚体(745.148m / z)(图2)。此外,使用指南针数据分析工具生成MurNAc-6P((M-H)的提取离子色谱图(EIC)= 372.070m / z)。 MurNAc-6P的EIC曲线下面积(AUC)使用Prism 6程序确定,基线设置为30(图3)。结合标准曲线分析MurNAc-6P的EIC的AUC可以用于量化在ΔmurQ突变体中积累的MurNAc-6P的量(Borisova等人, ,2016)。


图1. WT和Δ突变细胞的可溶性提取物的HPLC-MS分析。基本峰色谱图BPC)显示出类似的代谢物模式,其差异可以通过计算差示离子谱(DS)来显现

图2.图1中19.8至22.7分钟的差示离子谱(DS)信号的质谱。该化合物通过其确切质量((MH))识别为MurNAc-6P, 观察到= 372.071m / z,理论值= 372.070m / z,)二聚物的精确质量(745.148m / z >)和消除产品(282.039 m / z )

图3. MurNAc-6P的提取离子色谱图(EIC)。 搜索MurNAc-6P的372.071 m / z 的EIC显示野生型(WT)细胞(中断的黑线)没有信号,但是对于ΔmurQ < / 突变细胞(蓝线;上图)。后一个信号曲线下面积(AUC)允许使用已知浓度的MurNAc-6P标准(下图)(Unsleber等人,2017)量化MurNAc-6P的量。

笔记

  1. 只能使用HPLC级化学品和超纯的Millipore水进行样品制备和质谱分析。
  2. 用Millipore水广泛洗涤细菌培养物,以消除LB肉汤培养基中盐和成分的污染
  3. 避免在洗涤期间细菌悬浮液涡旋以防止细胞裂解。
  4. 在-80℃下冻结细菌培养物,以改善玻璃珠随后的细胞破坏
  5. 在LC-MS测量之前,使用冰冷的丙酮有效沉淀胞质部分中剩余的蛋白质
  6. 以负离子模式进行MurNAc-6P的检测。在正离子模式下无法检测到MurNAc-6P。

食谱

  1. 中等Luria-Bertani(LB)肉汤,米勒
    5克酵母提取物
    10克胰蛋白胨
    10g氯化钠
    在1升蒸馏水中 高压灭菌介质在121°C 15分钟
  2. LC-MS校准物(10mM甲酸钠)
    12.5毫升Millipore水
    12.5毫升丙-2-醇
    50微升甲酸
    250μl1N NaOH
  3. HPLC缓冲液A:0.1%甲酸,0.05%甲酸铵(pH3.2) 1毫升甲酸
    0.5g甲酸铵
    1升高压灭菌的Millipore水

致谢

该协议由Borisova等人(2016)进行了改编。 Christoph Mayer得到德意志银行(DFG)授权MA2436 / 7,SFB766 / A15和GRK1708 / B2的支持。

参考

  1. Borisova,M.,Gaupp,R.,Duckworth,A.,Schneider,A.,Dalugge,D.,Mühleck,M.,Deubel,D.,Unsleber,S.,Yu,W.,Muth, Bischoff,M.,Götz,F.和Mayer,C.(2016)。&nbsp; 革兰阳性菌中的肽聚糖回收对于稳定期的生存至关重要。 7(5)。
  2. Jaeger,T.,Arsic,M.和Mayer,C。(2005)。通过大肠杆菌“醚酶”分离出N-乙酰谷氨酸的乳糖醚键。 280(34):30100-30106。
  3. Litzinger,S.,Duckworth,A.,Nitzsche,K.,Risinger,C.,Wittmann,V. and Mayer,C。(2010)。枯草芽孢杆菌中的神经肽拯救涉及β-氨基葡糖苷酶的顺序水解, N-乙酰月桂酰-L-丙氨酸酰胺酶。 J Bacteriol 192(12):3132-3143。
  4. Reith J.和Mayer,C.(2011)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/21796380”target =“_ blank” >革兰氏阳性菌中的肽聚糖周转和回收利用。 Appl Microbiol Biotechnol 92(1):1-11。
  5. Uehara,T.,Suefuji,K.,Jaeger,T.,Mayer,C.and Park,JT(2006)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm .nih.gov / pubmed / 16452451“target =”_ blank“>大肠杆菌所要求的MurQ醚酶为了代谢获得的脱氢-NH-乙酰木瓜霉糖酸环境或其自身的细胞壁。 J Bacteriol 188(4):1660-1662。
  6. unsleber,S.,Borisova,M.和Mayer,C.(2017)。&nbsp; 酶的合成和半制备型分离的N-乙酰谷氨酸6-磷酸酯。碳水化合物445:98-103。
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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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