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Bacterial Porphyrin Extraction and Quantification by LC/MS/MS Analysis
采用 LC/MS/MS 分析法提取和量化细菌卟啉   

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Abstract

Heme is an iron-containing porphyrin which acts as a prosthetic group in several enzymes involved in disparate functions, such as respiration and H2O2-scavenging. Escherichia coli is able to produce heme endogenously since it contains all the enzymes involved in the nine-step biosynthesis pathway, which in absence of stress and in iron-replete media proceeds unabated. However, we recently showed that two steps are affected by H2O2 stress (Mancini and Imlay, 2015). To compensate, two enzymes, namely the ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF), are activated by the H2O2-responsive regulator OxyR. Genetic mutations that block either adaptation cause the intracellular accumulation of protoporphyrin IX and coproporphyrinogen III, the substrates of HemH and HemF, respectively. We here describe a method used to extract and quantify protoporphyrin IX and coproporphyrin III, the product of the spontaneous oxidation of coproporphyrinogen III.

Keywords: Heme(血红素), Protoporphyrin(原卟啉), Protoporphyrinogen(原卟啉原), Ferrochelatase(亚铁螯合酶)

Materials and Reagents

  1. Bacterial cell culture
  2. Ethyl acetate (Sigma-Aldrich, catalog number: 34972-1 L-R )
  3. Acetic acid (Sigma-Aldrich, catalog number: 45754-100 ML-F )
  4. Hydrochloric acid (Sigma-Aldrich, catalog number: H1758-100 ML )
  5. Protoporphyrin IX (Frontier Scientific, catalog number: P562-9 )
  6. Coproporphyrin III dihydrochloride (Frontier Scientific, catalog number: C-654-3 )
  7. Formic acid (Sigma-Aldrich, catalog number: 14265-1ML )
  8. Acetonitrile (Sigma-Aldrich, catalog number: 34967-250 ML )

Equipment

For porphyrin extraction

  1. Sonicator (Fisher Scientific, model: 550 sonic dismembrator )

For the LC/MS/MS analysis

  1. 5500 QTRAP LC/MS/MS system (AB Sciex)
  2. 1200 series HPLC system (Agilent Technologies)
  3. Degasser Autosampler (Agilent Technologies)
  4. Binary pump (Agilent Technologies)
  5. Zorbax SB-Aq column (4.6 x 50 mm, 5 µm) (Agilent Technologies, catalog number: 846975 )

Procedure

For the porphyrin extraction (adapted from Nakayashiki and Inokuchi, 1997)

  1. Culture bacterial cells until a final cell suspension equivalent to 100 ml with an A600 of 0.4.
  2. Harvest the cells by centrifugation at 7,000 x g at 4 °C for 10 min and wash the pellet in 20 ml pre-chilled 0.05 M Tris pH 8.2-2 mM EDTA.
  3. Resuspend the cells in 10 ml of the same buffer.
  4. Measure the A600 of the culture suspension and adjust to equalize the A600 of each sample by adding Tris-EDTA buffer.
  5. Spin down cells at 7,000 x g at 4 °C for 10 min. Resuspend the cell pellets in 1 ml ethyl acetate/acetic acid (3:1, v/v).
  6. Lyse the cells by sonication for 2 min with power 3 on ice.
  7. Remove the cell debris by centrifugation at 7,000 x g at 4 °C for 10 min and then transfer supernatant to a fresh tube.
  8. Add 1 ml H2O, vortex, and centrifuge at 7,000 x g for 5 min. Remove and discard the aqueous top layer.
  9. Repeat step 8.
  10. Add 100 µl of 3 M HCl to solubilize the porphyrins.
  11. Centrifuge after vigorous vortexing before transferring upper layer containing the water-soluble porphyrins into a fresh Eppendorf tube.

For the LC/MS/MS analysis

  1. Perform the LC separation with Zorbax SB-Aq column.
    1. Use 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B.
    2. Use a flow rate of 0.3 ml/min.
    3. Perform a stepwise elution as follows: 0-1 min 100% A, then gradually decrease down to 5% A until min 10. Maintain 5% A until min 18 and then increase to 100% A until min 19. Maintain 100% A until the end (min 24).
    4. Set the autosampler at 5 °C.
    5. Inject sample (1 μl) from step 11 above.
  2. Acquire the mass spectra with positive electrospray ionization (ESI) with the ion spray voltage set at 5,500 V.
    1. Set the source temperature at 450 °C.
    2. Set the curtain gas, ion source gas 1, and ion source gas 2 at 32, 65, and 50, respectively.
    3. Use multiple reactions monitoring (MRM) to monitor coproporphyrin III (m/z 655.4 --> m/z 596.3) and protoporphyrin IX (m/z 563.2 --> m/z 504.1). Use 1 µg of the porphyrin standards for the initial tests.
  3. Measure the peak areas of the resulting chromatograms with Analyst 1.6.
    Note that this is a qualitative assessment. A standard curve using the corresponding porphyrin references is required to obtain absolute values.

Representative data



Figure 1. MRM chromatograms of protoporphyrin IX. Intracellular accumulation of protoporphyrin IX in H2O2-stressed cells (Hpx2-) is exacerbated by the lack of hemH activation [Hpx2- hemH(NI)]. 1 µg protoporphyrin IX was included as reference standard.


Figure 2. MRM chromatograms of coproporphyrin III. Intracellular coproporphyrinogen III accumulates in H2O2-stressed cells lacking hemF (Hpx2- ∆hemF), with levels comparable to those detected in cells lacking both the coproporphyrinogen III oxidase isozymes (∆hemN ∆hemF). 1 µg coproporphyrin III was included as reference standard.

Notes

Upon exposure to air all porphyrinogens autoxidize to porphyrins. Therefore, coproporphyrin III and not coproporphyrinogen III (the actual substrate of the two coproporphyrinogen III oxidase isozymes HemN and HemF) can be analyzed and quantified by LC/MS/MS. Similarly, protoporphyrinogen IX, the substrate of the penultimate enzyme of the heme biosynthesis pathway, namely protoporphyrinogen IX oxidase, spontaneously oxidizes to protoporphyrin IX. Therefore, protoporphyrin IX levels should reflect the sum of protoporphyrinogen IX and protoporphyrin IX.

Acknowledgments

This work was supported by grant GM49640 from the National Institutes of Health and by award number PBBEP3_139397 from the Swiss National Science Foundation. The protocol for the porphyrin extraction was adapted from Nakayashiki and Inokuchi (1997).

References

  1. Mancini, S. and Imlay, J. A. (2015). The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress. Mol Microbiol 96(4): 744-763.
  2. Nakayashiki, T. and Inokuchi, H. (1997). Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli. Mol Gen Genet 255(4): 376-381.

简介

血红素是含铁的卟啉,其在参与不同功能(例如呼吸和H 2 O 2 O 2 - 清除)的几种酶中充当辅基。大肠杆菌能够内源性产生血红素,因为其含有涉及九步生物合成途径的所有酶,其在没有应激的情况下和在铁补充培养基中不减弱地进行。然而,我们最近显示两个步骤受H 2 O 2 O 2胁迫的影响(Mancini和Imlay,2015)。为了补偿,两种酶,即铁螯合酶(HemH)和粪卟啉原III氧化酶(HemF)的同工酶被H 2 O 2反应性调节剂OxyR激活。阻断任一适应的遗传突变导致原卟啉IX和粪卟啉原III的细胞内积累,分别是HemH和HemF的底物。我们在这里描述一种方法,用于提取和量化原卟啉IX和粪卟啉III,coproporphyrinogen III的自发氧化的产物。

关键字:血红素, 原卟啉, 原卟啉原, 亚铁螯合酶

材料和试剂

  1. 细菌细胞培养
  2. 乙酸乙酯(Sigma-Aldrich,目录号:34972-1L-R)
  3. 乙酸(Sigma-Aldrich,目录号:45754-100ML-F)
  4. 盐酸(Sigma-Aldrich,目录号:H1758-100ML)
  5. 原卟啉IX(Frontier Scientific,目录号:P562-9)
  6. 粪卟啉III二氢氯化物(Frontier Scientific,目录号:C-654-3)
  7. 甲酸(Sigma-Aldrich,目录号:14265-1ML)
  8. 乙腈(Sigma-Aldrich,目录号:34967-250ML)

设备

对于卟啉提取

  1. 超声波仪(Fisher Scientific,型号:550声波破碎仪)

对于LC/MS/MS分析

  1. 5500 QTRAP LC/MS/MS系统(AB Sciex)
  2. 1200系列HPLC系统(Agilent Technologies)
  3. 脱气自动进样器(Agilent Technologies)
  4. 二元泵(Agilent Technologies)
  5. Zorbax SB-Aq柱(4.6×50mm,5μm)(Agilent Technologies,目录号:846975)

程序

对于卟啉提取(改编自Nakayashiki和Inokuchi,1997)

  1. 培养细菌细胞,直至最终细胞悬浮液相当于100ml,A 600为0.4
  2. 通过在4℃下以7000xg离心10分钟收获细胞 并在20ml预冷的0.05M Tris pH 8.2-2mM EDTA中洗涤沉淀
  3. 将细胞重悬在10ml相同的缓冲液中。
  4. 测量培养悬浮液的A 600,并通过加入Tris-EDTA缓冲液调节以平衡每个样品的A 600。
  5. 在4℃下以7,000×g离心细胞10分钟。将细胞沉淀重悬在1ml乙酸乙酯/乙酸(3:1,v/v)中
  6. 在冰上用电源3超声处理细胞2分钟以裂解细胞
  7. 通过在4℃下以7000xg离心10分钟除去细胞碎片,然后将上清液转移到新管中。
  8. 加入1ml H 2 O,涡旋,并以7000xg离心5分钟。取出并丢弃含水顶层。
  9. 重复步骤8.
  10. 加入100μl的3M盐酸溶解卟啉
  11. 在转移上层之前,在剧烈涡旋之后离心 ?含有水溶性卟啉到新鲜的Eppendorf管中

对于LC/MS/MS分析

  1. 用Zorbax SB-Aq色谱柱进行LC分离
    1. 使用0.1%甲酸的水溶液作为流动相A和0.1%甲酸的乙腈溶液作为流动相B.
    2. 使用0.3ml/min的流速。
    3. 进行如下逐步洗脱:0-1分钟100%A,然后 逐渐降低到5%A,直到最小10.保持5%A,直到最小18 ?然后增加到100%A直到最小19.保持100%A直到 结束(分钟24)。
    4. 将自动进样器设置在5°C。
    5. 从上述步骤11注入样品(1μl)。
  2. 用正电喷雾电离(ESI)获得质谱,离子喷雾电压设置为5,500 V.
    1. 将源温度设置为450°C
    2. 将窗帘气体,离子源气体1和离子源气体2分别设置为32,65和50
    3. 使用多反应监测(MRM)监测粪卟啉 III(m/z 655.4-> m/z 596.3)和原卟啉IX(m/z 563.2-> ?m/z 504.1)。使用1μg卟啉标准品进行初始测试。
  3. 使用Analyst 1.6测量所得色谱图的峰面积。
    请注意,这是一个定性评估。使用标准曲线 需要相应的卟啉参考以获得绝对 值。

代表数据



图1.原卟啉IX的MRM色谱图。原卟啉IX在H 2 O 2亚型应激细胞中的细胞内积累(Hpx2 )由于缺少hemH 激活而加剧[Hpx2 - hemH (NI)]。包括1μg原卟啉IX作为参考标准

图2.粪卟啉III的MRM色谱图。细胞内粪卟啉原III在缺乏hemF - ΔhemF),其水平与在缺乏粪卟啉原III氧化酶同功酶(ΔhemNΔhemF)的细胞中检测到的水平相当。包括1μg粪卟啉III作为参照标准。

笔记

暴露于空气中时,所有卟啉原自动氧化成卟啉。因此,可以通过LC/MS/MS分析和定量粪卟啉III和非粪卟啉原III(两种粪卟啉原III氧化酶同工酶HemN和HemF的实际底物)。类似地,原卟啉原IX,血红素生物合成途径的倒数第二个酶的底物,即原卟啉原IX氧化酶,自发地氧化成原卟啉IX。因此,原卟啉IX水平应反映原卟啉原IX和原卟啉IX的总和。

致谢

这项工作得到国家卫生研究院授予GM49640和来自瑞士国家科学基金会的奖号PBBEP3_139397的支持。卟啉提取的方案改编自Nakayashiki和Inokuchi(1997)。

参考文献

  1. Mancini,S。和Imlay,J.A。(2015)。 两种生物合成酶的诱导有助于大肠杆菌维持血红素合成和激活过氧化氢酶在过氧化氢胁迫过程中的作用。 Mol Microbiol 96(4):744-763。
  2. Nakayashiki,T。和Inokuchi,H。(1997)。 饥饿对血红素对大肠杆菌中卟啉合成的影响。 Mol Gen Genet 255(4):376-381。
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引用:Mancini, S. and Imlay, J. A. (2015). Bacterial Porphyrin Extraction and Quantification by LC/MS/MS Analysis. Bio-protocol 5(19): e1616. DOI: 10.21769/BioProtoc.1616.
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