Stable-isotope Labeled Metabolic Analysis in Drosophila melanogaster: From Experimental Setup to Data Analysis
黑腹果蝇的稳定同位素标记代谢分析:从实验建立到数据分析   

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Abstract

Stable-isotope labeled metabolic analysis is an essential methodology to characterize metabolic regulation during biological processes. However, the method using stable-isotope-labeled tracer (e.g., 13C-glucose) in live animal is only beginning to be developed. Here, we contribute a qualitative metabolic labeling experiment protocol in Drosophila melanogaster using stable-isotope-labeled 13C-glucose tracer followed by liquid chromatography-mass spectrometry (LC-MS) analysis. Detailed experimental setup, data acquisition and analysis are provided to facilitate the application of in vivo metabolic labeling analysis that might be applied in a wide range of biological studies.

Keywords: Stable-isotope labeling (稳定同位素标记), 13C-glucose tracer (13C-葡萄糖示踪), Metabolic analysis (代谢分析), Qualitative analysis (定性分析), Liquid chromatography-mass spectrometry (液相色谱-质谱联用), Drosophila melanogaster (黑腹果蝇)

Background

Metabolomics is a newly emergent omic-level study aiming to profile small molecule metabolites in a complex biological system. It has been applied in diverse research areas pertaining to human health and disease, such as biomarker discovery, disease pathogenesis, and assessment of drug toxicity. Measurement of metabolites is important to determine alterations in metabolic pathway in response to endogenous and exogenous changes. To accurately characterize metabolic pathway activity, isotope-labeled tracers (e.g., 13C and 15N) have been used (Park et al., 2016; Jang et al., 2018). There are many such studies (both quantitatively and qualitatively) in cultured cells (Buescher et al., 2015; Liu et al., 2018), however, stable-isotope based metabolic labeling experiment in live animal remain largely unexplored. In the current protocol, we describe a qualitative metabolic labeling analysis by using the labeled 13C-glucose as a tracer, and we have successfully applied this protocol to comparatively analyze the activity of glycolysis pathway in Drosophila melanogaster, during aging and between wild-type and mutant animals.

Materials and Reagents

  1. Consumables
    1. Pipette tips (Eppendorf, catalog number: 0030073428 )
    2. Ceramic beads (Aoran, catalog number: 150010C )
    3. Eppendorf tube (2 ml) (Eppendorf, catalog number: 0030120094 )
    4. HPLC glass vial (Agilent Technologies, catalog number: 5182-0716 )
    5. Injection needle (Agilent Technologies, catalog number: G4226-87201 )
    6. Kimwipe filter paper (KCWW, Kimberly-Clark, catalog number: 34120 )

  2. Biological material
    1. Drosophila melanogaster
      The Drosophila strain used was 5905 (FlyBase ID: FBst0005905, w1118). Flies were cultured in standard media (Recipe 1) at 25 °C with 60% humidity in a 12 h light and 12 h dark cycle.
      Prior to the test, flies were starved on 1% Agar media for 6 h before transferred to the vials containing a small piece of Kimwipe filter paper (KCWW, Kimberly-Clark, catalog number: 34120 ) pre-soaked in 1 ml of 10% U-13C6-glucose (U-13C6-glucose was added to phosphate buffer at a final concentration of 10%). Flies were treated for 3 days, and then transferred to new vials with fresh U-13C6-glucose for additional 2 days. Fly heads were dissected from anesthetized flies with CO2 for subsequent metabolic analysis. For each experiment, 8 biological repeats were conducted, with 20 heads for each repeat. One hundred and sixty male flies were used, with 20 flies per vial.

  3. Chemicals
    LC-MS chemicals:
    1. Methanol (MeOH), LC-MS grade (Honeywell, catalog number: LC230-2.5HC ). Store at the room temperature (20 °C-25 °C)
    2. Acetonitrile (ACN), LC-MS grade (Merck, catalog number: 1.00029.2500 ). Store at the room temperature (20 °C-25 °C)
    3. Water (H2O) (Honeywell, catalog number: LC365-2.5HC ). Store at the room temperature (20 °C-25 °C)
    4. Ammonium acetate, LC-MS grade (Sigma-Aldrich, catalog number: 73594-25G-F ). Store at 4 °C
    5. Ammonium hydroxide, LC-MS grade (Sigma-Aldrich, catalog number: 44273-100mL-F ). Store at 4 °C
    6. Liquid nitrogen

    Labeled chemicals:
    1. D-Glucose (U-13C6, 99%) (Cambridge Isotope Laboratories, catalog number: CLM-1396-PK ). Store at the room temperature (20 °C-25 °C)

    Drosophila standard media:
    1. Sucrose 
    2. Maltose 
    3. Yeast 
    4. Agar 
    5. Maizena 
    6. Soybean flour
    7. 438 sodium benzoate 
    8. Methyl-p-hydroxybenzoate
    9. Propionic acid

  4. Mobile phase setup
    1. Mobile phase A (see Recipes)
    2. Mobile phase B (see Recipes)

Equipment

  1. Pipettes
  2. Homogenizer (BERTIN, model: Precellys® 24 )
  3. Incubator
  4. Sonicator
  5. Centrifuge
  6. Vacuum concentrator (Labconco, German)
  7. Merck SeQuant ZIC-pHILIC column [particle size, 5 μm; 100 mm (length) x 2.1 mm (i.d.)]
  8. UHPLC system (Agilent Technologies, model: 1290 Infinity )
  9. Quadruple time-of-flight mass spectrometer (Agilent Technologies, model: 6550 Series )

Software

  1. Pathways to PCDL (version B.07.00, Agilent Technologies)
  2. PCDL Manager (version B.07.00, Agilent Technologies)
  3. Profinder (version B.08.00, Agilent Technologies)
  4. MassHunter software (version B.07.00, Agilent Technologies)

Procedure

  1. Metabolites extraction
    1. Quickly freeze the animal tissues (head of Drosophila) in liquid nitrogen immediately after dissection.
    2. Homogenize the tissue sample with 200 μl of H2O and 5 ceramic beads using the homogenizer.
    3. Add 800 μl ACN:MeOH (1:1, v/v) to homogenized solution for subsequent metabolite extraction.
    4. Incubate the samples for 1 h at -20 °C to precipitate proteins.
    5. Proceed with 15 min centrifugation at 15,000 x g under 4 °C.
    6. Transfer the resulting supernatant to a new Eppendorf tube (2 ml), then evaporate to dryness in a vacuum concentrator under 4 °C.
    7. Reconstitute the dry extracts with 100 µl of ACN:H2O (1:1, v/v).
    8. Sonicate the reconstitution solution for 10 min, and centrifuge for 15 min at 15,000 x g under 4 °C to remove insoluble debris.
    9. Transfer the supernatant to an HPLC glass vial and store at -80 °C if the samples will be subjected to LC-MS analysis within 3 h. For extracted samples that require long time (over 12 h) stored prior to being analyzed, we suggest storing the samples after Step A6 and then proceeding with A8-A9 before LC-MS analysis.

  2. LC-MS analysis
    1. Liquid chromatography
      1. Load worklist with method embedded using MassHunter software. Please note that LC-MS operation (both instrument and software) requires specialized training.
      2. Run batch sequence with following LC parameters:
        1. Wash injection needle one time with needle washing solvent MeOH:H2O (1:1, v/v).
        2. Load sample and inject 2 μl of sample.
        3. Run LC method using the LC gradient as described in Table 1.

          Table 1. The gradient elution method for LC-MS analysis


    2. Mass spectrometry
      Set MS parameters as described below:
      1. ESI source parameters:
        1. Sheath gas temperature, 300 °C.
        2. Dry gas temperature, 250 °C.
        3. Sheath gas flow, 12 L/min.
        4. Dry gas flow, 16 L/min.
        5. Capillary voltage, 2,500 V (+) and -2,500 V (-), respectively. Please note that the same sample is analyzed twice for each ionization mode.
        6. Nozzle voltage, 0 V.
        7. Nebulizer pressure, 20 psi.
      2. Time of Flight (TOF) parameters:
        1. TOF scan range: m/z 60-1,200 Da.
        2. MS1 acquisition frequency: 4 Hz.

Data analysis

  1. Extraction of isotopologues
    1. Metabolite library construction
      Use Pathways to PCDL software (version B.07.00, Agilent Technologies) and PCDL Manager software (version B.07.00, Agilent Technologies) to build a metabolite library for metabolites in both glycolysis and citric acid cycle. Specifically, each metabolite standard is analyzed under the same LC-MS condition as biological samples. The ion chromatograph of each metabolite is extracted to obtain the retention time information. Then, the retention time together with formula value is used to construct a metabolite library using PCDL manager. The input example is provided as below (Table 2):

      Table 2. The metabolite library for metabolites in both glycolysis and citric acid cycle


    2. Raw data loading
      Load the acquired LC-MS raw data files (.d) into Profinder (version B.08.00, Agilent Technologies) for the extraction of metabolite isotopologues using the constructed metabolite library.
    3. Feature extraction parameters:
      1. Ion abundance criterion: peak core area 20% of peak height.
      2. Mass tolerance: ± 15 ppm + 2.00 mDa.
      3. Retention time tolerance: ± 0.20 min.
      4. Anchor ion height threshold: 250 counts.
      5. Sum of ion heights threshold: 1,000 counts.
      6. Correlation coefficient threshold: 0.5.

  2. Stable-isotope-labeled metabolic analysis
    1. Peak integration result manual check
      After isotopologues extraction in Profinder, peak integration result need to be reviewed and manually curated for subsequent accurate stable-isotope labeled metabolic analysis. Make sure the peak integration range is consistent across multiple samples. Figure 1A illustrates the extracted ion chromatography (EIC) of the key metabolite 13C3-lactate and the peak integration range.
    2. Calculation of tracer incorporation
      For each targeted metabolite, different isotope pattern will be obtained corresponding to the number of incorporated 13C atoms. For example, the isotopologues of lactate are m + 0, m + 1, m + 2, and m + 3 (Figure 1B).
      1. Abundance of individual isotopologue is the integrated peak area.
        Taken metabolite lactate as an example, Figure 1 C shows the abundance level of one isotopologue of lactate M + 3 between two groups (wild type and PRC2 mutant). In the article by Ma et al., 2018, Figure 6G is generated using this calculated data.
      2. Total metabolite abundance
        Total metabolite abundance is calculated using the following formula:



        Mn is the labeling pattern of the isotopologue with all atoms (C or N) labeled.
        Figure 1 D shows the total abundance level of lactate between two groups (wild type and PRC2 mutant). In the article by Ma et al., 2018, Figure S5E was generated using this calculated data.
      3. Proportion of individual isotopologue
        Proportion of individual isotopologue is calculated using the following formula:



        Mi is the labeling pattern of individual isotopologue.
        Mn is the labeling pattern of the isotopologue with all atoms (C or N) labeled.
        Figure 1 E shows the percentage of total pool level of one isotopologue of lactate M + 3 between two groups (wild type and PRC2 mutant). In the article by Ma et al., 2018, Figure S5B and Figure S5D were generated using this calculated data.
      4. Total tracer incorporation
        Total tracer incorporation is calculated using the following formula:



        Figure 1F shows the percentage of total tracer incorporation between two groups (wild type and PRC2 mutant).
        Above results demonstrated that lactate, the end product of glycolysis pathway, significantly increased in PRC2 mutants.


        Figure 1. Stable-isotope-labeled metabolic analysis strategy. A. The extracted ion chromatography (EIC) of isotopologue 13C3-lactate (m + 3). (mean ± SD of 8 biological repeats with 10 flies for each measurement; Student's t-test; n.s.: not significant). Test was from muscle tissues of 30 d old male flies. Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12c253/+. B. The labeling pattern of lactate demonstrated in mass spectrum (m + 0, m + 1, m + 2, and m + 3). C. The abundance level of lactate isotopologue m + 3 between two groups (mean ± SD of 8 biological repeats with 10 flies for each measurement; Wilcox test). Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12c253/+. D. The total abundance level of lactate between two groups (wild type and PRC2 mutant) (mean ± SD of 8 biological repeats with 10 flies for each measurement; Wilcox test). Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12c253/+. E. The percentage of total pool level of one isotopologue of lactate m + 3 between two groups (mean ± SD of 8 biological repeats with 10 flies for each measurement; Wilcox test). Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12c253/+. F. The percentage of total tracer incorporation between two groups (mean ± SD of 8 biological repeats with 10 flies for each measurement; Wilcox test). Genotypes: WT: 5905. Mut: Pclc421/+; Su(z)12c253/+.

Recipes

  1. Standard Drosophila food
    Sucrose 36 g/L 
    Maltose 38 g/L 
    Yeast 22.5 g/L 
    Agar 5.4 g/L 
    Maizena 60 g/L 
    Soybean flour 8.25 g/L 
    438 sodium benzoate 0.9 g/L 
    Methyl-p-hydroxybenzoate 0.225 g/L
    Propionic acid 6.18 ml/L
    ddH2O to make up 1 L
  2. Mobile phase A
    25 mM ammonium acetate
    25 mM ammonium hydroxide
    For the preparation of 1 L mobile phase A, firstly weigh 1.9271 g CH3COONH4. Dissolve the CH3COONH4 in 1 L H2O. Then add 3.5 ml NH4OH (25%) to generate the mobile phase A. Store the solution at 4 °C for up to 2 weeks
  3. Mobile phase B
    Acetonitrile
    Store at the room temperature (20-25 °C)

Acknowledgments

We thank the financial support provided by the startup funding from Interdisciplinary Research Center on Biology and Chemistry (IRCBC), and Agilent Technologies Thought Leader Award. N.L. and Z.-J. Z. are also supported by Thousand Youth Talents Program. This protocol is also a part of our previous work by Ma et al., 2018.

Competing interests

The authors declare no competing financial interest.

References

  1. Buescher, J. M., Antoniewicz, M. R., Boros, L. G., Burgess, S. C., Brunengraber, H., Clish, C. B., DeBerardinis, R. J., Feron, O., Frezza, C., Ghesquiere, B., Gottlieb, E., Hiller, K., Jones, R. G., Kamphorst, J. J., Kibbey, R. G., Kimmelman, A. C., Locasale, J. W., Lunt, S. Y., Maddocks, O. D., Malloy, C., Metallo, C. M., Meuillet, E. J., Munger, J., Noh, K., Rabinowitz, J. D., Ralser, M., Sauer, U., Stephanopoulos, G., St-Pierre, J., Tennant, D. A., Wittmann, C., Vander Heiden, M. G., Vazquez, A., Vousden, K., Young, J. D., Zamboni, N. and Fendt, S. M. (2015). A roadmap for interpreting 13C metabolite labeling patterns from cells. Curr Opin Biotechnol 34: 189-201.
  2. Jang, C., Chen, L. and Rabinowitz, J. D. (2018). Metabolomics and isotope tracing. Cell 173(4): 822-837.
  3. Liu, L., Su, X., Quinn, W. J., 3rd, Hui, S., Krukenberg, K., Frederick, D. W., Redpath, P., Zhan, L., Chellappa, K., White, E., Migaud, M., Mitchison, T. J., Baur, J. A. and Rabinowitz, J. D. (2018). Quantitative analysis of NAD synthesis-breakdown fluxes. Cell Metab 27(5): 1067-1080 e1065.
  4. Ma, Z., Wang, H., Cai, Y., Wang, H., Niu, K., Wu, X., Ma, H., Yang, Y., Tong, W., Liu, F., Liu, Z., Zhang, Y., Liu, R., Zhu, Z. J. and Liu, N. (2018). Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila. Elife 7: e35368.
  5. Park, J. O., Rubin, S. A., Xu, Y. F., Amador-Noguez, D., Fan, J., Shlomi, T. and Rabinowitz, J. D. (2016). Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. Nat Chem Biol 12(7): 482-489.

简介

稳定同位素标记的代谢分析是表征生物过程中代谢调节的基本方法。 然而,在活体动物中使用稳定同位素标记的示踪剂(例如, 13 C-葡萄糖)的方法才刚刚开始开发。 在这里,我们使用稳定同位素标记的 13 C-葡萄糖示踪剂,然后通过液相色谱 - 质谱(LC-MS)在 Drosophila melanogaster 中提供定性代谢标记实验方案。 分析。 提供详细的实验设置,数据采集和分析以促进体内代谢标记分析的应用,其可以应用于广泛的生物学研究中。

【背景】代谢组学是一项新兴的omic-level研究,旨在研究复杂生物系统中的小分子代谢物。它已被应用于与人类健康和疾病有关的各种研究领域,例如生物标志物发现,疾病发病机理和药物毒性评估。代谢物的测量对于确定响应内源和外源变化的代谢途径的改变是重要的。为了准确表征代谢途径活动,已使用同位素标记的示踪剂(例如, 13 C和 15 N)(Park et al。,2016; Jang et al。,2018)。在培养的细胞中有许多这样的研究(定量和定性)(Buescher et al。,2015; Liu et al。,2018),然而,基于稳定同位素的研究活体动物的代谢标记实验在很大程度上尚未开发。在目前的方案中,我们描述了使用标记的 13 C-葡萄糖作为示踪剂的定性代谢标记分析,并且我们已成功应用该方案来比较分析中的糖酵解途径的活性。果蝇(Drosophila melanogaster),在衰老期间以及野生型和突变体动物之间。

关键字:稳定同位素标记, 13C-葡萄糖示踪, 代谢分析, 定性分析, 液相色谱-质谱联用, 黑腹果蝇

材料和试剂

  1. 耗材
    1. 移液器吸头(Eppendorf,目录号:0030073428)
    2. 陶瓷珠(Aoran,目录号:150010C)
    3. Eppendorf管(2毫升)(Eppendorf,目录号:0030120094)
    4. HPLC玻璃瓶(Agilent Technologies,目录号:5182-0716)
    5. 注射针(Agilent Technologies,目录号:G4226-87201)
    6. Kimwipe滤纸(KCWW,Kimberly-Clark,目录号:34120)

  2. 生物材料
    1. Drosophila melanogaster
      使用的 Drosophila 菌株是5905(FlyBase ID:FBst0005905, w 1118 )。将蝇在标准培养基(配方1)中在25℃,60%湿度,12小时光照和12小时黑暗循环中培养。
      在测试之前,将苍蝇在1%琼脂培养基上饥饿6小时,然后转移到含有一小片Kimwipe滤纸(KCWW,Kimberly-Clark,目录号:34120)的小瓶中,预先浸泡在1ml 10%中。将U- 13 C 6 - 葡萄糖(U- 13 C 6 - 葡萄糖加入到磷酸盐缓冲液中最终浓度为10%)。将果蝇处理3天,然后转移到具有新鲜U- 13 C 6 - 葡萄糖的新小瓶中另外2天。从具有CO 2 的麻醉苍蝇中解剖蝇头用于随后的代谢分析。对于每个实验,进行8次生物重复,每次重复20个头。使用了160只雄性果蝇,每瓶20只苍蝇。

  3. 化学品
    LC-MS化学品:
    1. 甲醇(MeOH),LC-MS级(Honeywell,目录号:LC230-2.5HC)。在室温下储存(20°C-25°C)
    2. 乙腈(ACN),LC-MS级(Merck,目录号:1.00029.2500)。在室温下储存(20°C-25°C)
    3. 水(H 2 O)(霍尼韦尔,目录号:LC365-2.5HC)。在室温下储存(20°C-25°C)
    4. 乙酸铵,LC-MS级(Sigma-Aldrich,目录号:73594-25G-F)。储存在4°C
    5. 氢氧化铵,LC-MS级(Sigma-Aldrich,目录号:44273-100mL-F)。储存在4°C
    6. 液氮

    标记化学品:
    1. D-葡萄糖(U- 13 C 6 ,99%)(Cambridge Isotope Laboratories,目录号:CLM-1396-PK)。在室温下储存(20°C-25°C)

    Drosophila 标准媒体:
    1. 蔗糖 
    2. 麦芽糖 
    3. 酵母 
    4. 琼脂 
    5. Maizena 
    6. 大豆粉
    7. 438苯甲酸钠 
    8. 甲基对羟基苯
    9. 丙酸

  4. 流动相设置
    1. 流动相A(见食谱)
    2. 流动相B(见食谱)

设备

  1. 移液器
  2. 均质器(BERTIN,型号:Precellys ® 24)
  3. 恒温箱
  4. 超声仪
  5. 离心机
  6. 真空浓缩器(Labconco,德国)
  7. Merck SeQuant ZIC-pHILIC色谱柱[粒径,5μm; 100毫米(长)x 2.1毫米(i.d.)]
  8. UHPLC系统(Agilent Technologies,型号:1290 Infinity)
  9. 四倍飞行时间质谱仪(Agilent Technologies,型号:6550系列)

软件

  1. 通往PCDL的途径(版本B.07.00,安捷伦科技)
  2. PCDL Manager(版本B.07.00,安捷伦科技)
  3. Profinder(版本B.08.00,安捷伦科技)
  4. MassHunter软件(版本B.07.00,安捷伦科技)

程序

  1. 代谢物提取
    1. 解剖后立即快速冷冻液氮中的动物组织(果蝇头部)。
    2. 使用均化器用200μlH2 sub 2和5个陶瓷珠均化组织样品。
    3. 将800μlACN:MeOH(1:1,v / v)加入均质溶液中,用于随后的代谢物提取。
    4. 将样品在-20°C孵育1小时以沉淀蛋白质。
    5. 在4℃下在15,000 x g 下进行15分钟离心。
    6. 将得到的上清液转移到新的Eppendorf管(2ml)中,然后在真空浓缩器中在4℃下蒸发至干。
    7. 用100μlACN:H 2 O(1:1,v / v)重构干提取物。
    8. 超声处理重构溶液10分钟,并在4℃下以15,000 x g 离心15分钟以除去不溶性碎片。
    9. 将上清液转移到HPLC玻璃小瓶中并在-80℃下储存,如果样品将在3小时内进行LC-MS分析。对于在分析之前需要长时间(超过12小时)存储的提取样品,我们建议在步骤A6之后存储样品,然后在LC-MS分析之前继续进行A8-A9。

  2. LC-MS分析
    1. 液相色谱
      1. 使用MassHunter软件嵌入方法加载工作清单。请注意,LC-MS操作(仪器和软件)需要专门的培训。
      2. 使用以下LC参数运行批次序列:
        1. 用针头洗涤溶剂MeOH:H 2 O(1:1,v / v)洗涤注射针一次。
        2. 加载样品并注入2μl样品。
        3. 使用表1中所述的LC梯度运行LC方法。

          表1.用于LC-MS分析的梯度洗脱方法


    2. 质谱分析
      如下所述设置MS参数:
      1. ESI源参数:
        1. 鞘气温度,300°C。
        2. 干燥气体温度,250°C。
        3. 护套气流量,12 L / min。
        4. 干气流量,16 L / min。
        5. 毛细管电压分别为2,500 V(+)和-2,500 V( - )。请注意,每种电离模式都会对同一样品进行两次分析。
        6. 喷嘴电压,0 V.
        7. 雾化器压力,20 psi。
      2. 飞行时间(TOF)参数:
        1. TOF扫描范围:m / z 60-1,200 Da。
        2. MS1采集频率:4 Hz。

数据分析

  1. 同位素的提取
    1. 代谢物库建设
      使用Pathways to PCDL软件(版本B.07.00,安捷伦科技)和PCDL Manager软件(版本B.07.00,安捷伦科技),在糖酵解和柠檬酸循环中构建代谢物的代谢物库。具体地,在与生物样品相同的LC-MS条件下分析每种代谢物标准品。提取每种代谢物的离子色谱图以获得保留时间信息。然后,使用PCDL管理器将保留时间与公式值一起用于构建代谢物库。输入示例如下(表2):

      表2.糖酵解和柠檬酸循环中代谢物的代谢物库


    2. 原始数据加载
      将获得的LC-MS原始数据文件(.d)加载到Profinder(版本B.08.00,Agilent Technologies)中,使用构建的代谢物库提取代谢物同位素异构体。
    3. 特征提取参数:
      1. 离子丰度准则:峰值核心面积为峰高的20%。
      2. 质量公差:±15 ppm + 2.00 mDa。
      3. 保留时间公差:±0.20分钟。
      4. 锚定离子高度阈值:250个计数。
      5. 离子高度阈值总和:1,000计数。
      6. 相关系数阈值:0.5。

  2. 稳定同位素标记的代谢分析
    1. 峰积分结果手册检查
      在Profinder中提取同位素后,需要对峰积分结果进行评估并手动策划,以便随后进行准确的稳定同位素标记代谢分析。确保多个样品的峰积分范围一致。图1A说明了关键代谢物 13 C 3 -lactate的提取离子色谱(EIC)和峰积分范围。
    2. 示踪剂结合的计算
      对于每种靶向代谢物,将获得对应于掺入的 13 C原子数的不同同位素模式。例如,乳酸的同位素异构体是m + 0,m + 1,m + 2和m + 3(图1B)。
      1. 个别同位素的丰度是综合的峰面积。
        以代谢物乳酸盐为例,图1C显示两组(野生型和PRC2突变体)之间乳酸盐M + 3的一种同位素异构体的丰度水平。在Ma 等人的文章,2018中,使用该计算的数据生成图6G。
      2. 总代谢物丰度
        总代谢物丰度使用以下公式计算:



        Mn是同位素异构体的标记模式,标记了所有原子(C或N)。
        图1D显示两组(野生型和PRC2突变体)之间乳酸的总丰度水平。在Ma et al。,2018的文章中,使用该计算数据生成图S5E。
      3. 个别同位素的比例
        个体同位素的比例使用以下公式计算:



        M i 是个别同位素的标记模式。
        Mn是同位素异构体的标记模式,标记了所有原子(C或N)。
        图1E显示了两组(野生型和PRC2突变体)之间乳酸M + 3的一种同位素异构体的总池水平的百分比。在Ma 等人的文章中,使用该计算数据生成2018,图S5B和图S5D。
      4. 总跟踪器合并
        总示踪剂掺入量使用以下公式计算:



        图1F显示了两组(野生型和PRC2突变体)之间总示踪剂掺入的百分比。
        以上结果表明,在PRC2突变体中,糖酵解途径的终产物乳酸显着增加。


        图1.稳定同位素标记的代谢分析策略。 A.同位素 13 C 3 -lactate的提取离子色谱(EIC) m + 3)。 (8次生物重复的平均值±SD,每次测量10只苍蝇;学生 t - 测试; n.s。:不显着)。测试来自30日龄雄性果蝇的肌肉组织。基因型:WT:5905.Mut: Pcl c421 / +; 素(z)的 12 C253 / +。 B.乳酸的标记模式在质谱(m + 0,m + 1,m + 2和m + 3)中显示。 C.两组之间乳酸同位素m + 3的丰度水平(8次生物重复的平均值±SD,每次测量10只果蝇; Wilcox测试)。基因型:WT:5905.Mut: Pcl c421 / +; 素(Z)12 C253 / +。 D.两组(野生型和PRC2突变体)之间乳酸的总丰度水平(8次生物重复的平均值±SD,每次测量10只果蝇; Wilcox测试)。基因型:WT:5905.Mut: Pcl c421 / +; 素(Z)12 C253 / +。 E.两组之间乳酸盐m + 3的一种同位素组的总池水平的百分比(8次生物重复的平均值±SD,每次测量10只果蝇; Wilcox测试)。基因型:WT:5905.Mut: Pcl c421 / +; 素(Z)12 C253 / +。 F.两组之间总示踪剂掺入的百分比(8次生物重复的平均值±SD,每次测量10只果蝇; Wilcox测试)。基因型:WT:5905.Mut:Pclc421 / +; Su(z)12c253 / +。

食谱

  1. 标准 Drosophila 食物
    蔗糖(36克/升)
    麦芽糖(38克/升)
    酵母(22.5克/升)
    琼脂(5.4克/升)
    Maizena(60克/升)
    大豆粉(8.25克/升)
    438苯甲酸钠(0.9 g / L)
    对羟基苯甲酸甲酯(0.225g / L)
    丙酸(6.18 ml / L)
    ddH 2 O组成1升
  2. 流动阶段A
    25 mM醋酸铵
    25mM氢氧化铵
    为制备1L流动相A,首先称重1.9271g CH 3 COONH 4 。将CH 3 COONH 4 溶解在1L H 2 O中。然后加入3.5 ml NH 4 OH(25%)以产生流动相A.将溶液在4°C下储存长达2周
  3. 流动阶段B
    乙腈
    储存在室温(20-25°C)

致谢

我们感谢来自生物学和化学跨学科研究中心(IRCBC)和安捷伦科技思想领袖奖的启动资金提供的财务支持。 N.L.和Z.-J. Z.也得到了千人青年计划的支持。该协议也是我们之前Ma 等人,2018年的工作的一部分。

利益争夺

作者声明没有竞争性的经济利益。

参考

  1. Buescher,JM,Antoniewicz,MR,Boros,LG,Burgess,SC,Brunengraber,H.,Clish,CB,DeBerardinis,RJ,Feron,O.,Frezza,C.,Ghesquiere,B.,Gottlieb,E.,Hiller ,K.,Jones,RG,Kamphorst,JJ,Kibbey,RG,Kimmelman,AC,Locasale,JW,Lunt,SY,Maddocks,OD,Malloy,C.,Metallo,CM,Meuillet,EJ,Munger,J。, Noh,K.,Rabinowitz,JD,Ralser,M.,Sauer,U.,Stephanopoulos,G.,St-Pierre,J.,Tennant,DA,Wittmann,C.,Vander Heiden,MG,Vazquez,A., Vousden,K.,Young,JD,Zamboni,N。和Fendt,SM(2015)。 从细胞中解释 13 C代谢物标记模式的路线图。 Curr Opin Biotechnol 34:189-201。
  2. Jang,C.,Chen,L。和Rabinowitz,J。D.(2018)。 代谢组学和同位素示踪。 细胞 173(4) :822-837。
  3. Liu,L.,Su,X,Quinn,WJ,3rd,Hui,S.,Krukenberg,K.,Frederick,DW,Redpath,P.,Zhan,L.,Chellappa,K.,White,E., Migaud,M.,Mitchison,TJ,Baur,JA和Rabinowitz,JD(2018)。 NAD合成 - 分解通量的定量分析。 Cell Metab > 27(5):1067-1080 e1065。
  4. Ma,Z.,Wang,H.,Cai,Y.,Wang,H.,Niu,K.,Wu,X.,Ma,H.,Yang,Y.,Tong,W.,Liu,F., Liu,Z.,Zhang,Y.,Liu,R.,Zhu,ZJ和Liu,N。(2018)。 老化中H3K27me3的表观遗传漂移将糖酵解与果蝇中的健康长寿联系起来。 Elife 7:e35368。
  5. Park,J.O.,Rubin,S.A.,Xu,Y.F.,Amador-Noguez,D.,Fan,J.,Shlomi,T。和Rabinowitz,J.D。(2016)。 代谢物浓度,通量和自由能意味着有效的酶用量。 Nat Chem Biol 12(7):482-489。
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Copyright Cai 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. Cai, Y., Liu, N. and Zhu, Z. (2018). Stable-isotope Labeled Metabolic Analysis in Drosophila melanogaster: From Experimental Setup to Data Analysis. Bio-protocol 8(18): e3015. DOI: 10.21769/BioProtoc.3015.
  2. Ma, Z., Wang, H., Cai, Y., Wang, H., Niu, K., Wu, X., Ma, H., Yang, Y., Tong, W., Liu, F., Liu, Z., Zhang, Y., Liu, R., Zhu, Z. J. and Liu, N. (2018). Epigenetic drift of H3K27me3 in aging links glycolysis to healthy longevity in Drosophila. Elife 7: e35368.
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