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Feb 2020

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Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry
用气相色谱-质谱法追踪小鼠组织中的氮代谢   

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Abstract

Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis of amino acids and nucleotides. Both nuclear magnetic resonance spectroscopy and mass spectrometry including gas chromatography-mass spectrometry (GC MS) and liquid chromatography (LC MS) have been used to measure nitrogen metabolism. Here we describe a protocol to trace nitrogen metabolism in multiple mouse tissues using 15N-ammonia coupled with GC MS. This protocol includes detailed procedures in tracer injection, tissue preparation, metabolite extraction, GC MS analysis and natural abundance corrections. This protocol will provide a useful tool to study tissue-specific nitrogen in metabolically active tissues such as the retina, brain, liver, and tumor.

Keywords: GC MS (气质联用), Mass spectrometry (质谱分析法), Nitrogen metabolism (氮代谢), 15N tracing (15N示踪), Ammonia metabolism (氨代谢), Amino acids (氨基酸), Ammonia (氨), Stable isotope tracer (稳定同位素示踪剂)

Background

Nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry including mass spectrometry (GC MS) and liquid chromatography (LC MS) have been successfully used for system-wide metabolite measurements in various organisms (Fiehn, 2002 and 2016; Chokkathukalam et al., 2014). However, each method has its limitations depending on the type of studies, which include absolute quantification, metabolite properties, sensitivity, robustness, isotope analysis and cost-effectiveness (Chokkathukalam et al., 2014). Compared to NMR, MS-based approaches are more commonly used as a result of their higher sensitivity and metabolite coverage. Stable isotope labeling coupled with LC MS or GC MS allows for sensitively quantifying dynamic metabolic changes in healthy and diseased tissue or cells (Jang et al., 2018). LC MS coupled with stable nitrogen isotope reveals how glutamine nitrogen metabolism coordinates with carbon metabolism in cancer cells (Wang et al., 2019). GC MS has superior chromatographic resolution and cost-effectiveness (Jang et al., 2018). Here we have developed a robust method to quantify nitrogen-derived metabolites using stable nitrogen isotope coupled with GC MS. We identified several key metabolic features in the retina with this method, including the metabolic communications within the neural retina and between the retina and retinal pigment epithelium (RPE) (Albertino et al., 1989; Du et al., 2015 and 2016; Grenell et al., 2019; Yam et al., 2019). Recently, we used 15N-ammonia to trace nitrogen metabolism in vivo in mice and revealed tissue-specific metabolic pathways (Xu et al., 2020). In this protocol, we described procedures in nitrogen tracer injection in mice, tissue preparation, metabolite extraction, GC MS sample preparation, instrument analysis, and natural abundance corrections.


Materials and Reagents

  1. 2 ml microtube

  2. 1.5 ml tube

  3. Syringe with gauge 26 needle (BD, catalog number: 9304308)

  4. Syringe for 0.2 μm syringe filter (BD, catalog number: 7212651)

  5. ALS Syringe for GC injection (Agilent Technologies, catalog number: 5181-3354)

  6. Microtubes (Axygen Scientific, catalog number: 02820037 for 1.5 ml and catalog number: 12018028 for 2 ml)

  7. 0.2 μm syringe filter (Thermo Scientific, catalog number: 00293112)

  8. Cap for GC MS vial (Agilent Technologies, catalog number: 5182-0717)

  9. Glass inserts (Agilent Technologies, catalog number: 5181-2085, specification: 250 μl)

  10. Disposable pestle (Argos Technologies, catalog number: 7339-901)

  11. C57 B6/J mouse (Jackson Lab, catalog number: 664)

  12. Ethanol (Fisher Chemical, catalog number: 172382, LOT-specific concentration: 96%)

  13. Liquid nitrogen

  14. Methanol (Fisher Chemical, OptimaTM LC/MS Grade, catalog number: 164905)

  15. HPLC Water (Fisher Chemical, OptimaTM LC/MS Grade, catalog number:7732-18-5)

  16. Hexane (Sigma-Aldrich, HPLC Grade, catalog number: MKCF5755)

  17. Methylene Chloride (Fisher Chemical, catalog number: 163938)

  18. Helium Gas (MATHESON, catalog number: HE UHP1A)

  19. Pyridine (Sigma-Aldrich, catalog number: SHBK1583)

  20. N-tert-Butyldimethylsily-N-methyltrifluoroacetamide (TBDMS) (Sigma-Aldrich, catalog number: 394882)

  21. Ammonium-15N Chloride (15NH4Cl) (Sigma-Aldrich, catalog number: 229251)

  22. Amino Acid Standard mix (Sigma-Aldrich, catalog number: AAS18-5 ml)

  23. 10× Phosphate Buffered Saline (PBS) (Fisher Chemical, catalog number: 10010049)

  24. Hank's Balanced Salt Solutions (HBSS) (Fisher Chemical, catalog number: 14170112)

  25. EDTA (Fisher Chemical, catalog number: 6N011324)

  26. 15NH4Cl- solution (see Recipes)

  27. Extraction buffer (see Recipes)

  28. Internal Standard (see Recipes)

  29. Methoxyamine mix (see Recipes)

Equipment

  1. Dumont Tweezer, Style 5 (Electron Microscopy Sciences, catalog number: 0108-5-PO)

  2. -20 °C refrigerator

  3. Dissecting microscope (Zeiss, model: Stemi 2000-C)

  4. FirstHand Surgical Instrument Kits for Mice and Rats (Kent Scientific Corporation, model: INSMOUSEKIT)

  5. Battery-Operated Pestle Motor Mixer (Argos Technologies, catalog number: EW-44468-25)

  6. Omni Tissue Homogenizer (115V 125W) (OMNI International the Homogenizer Company, THB-01)

  7. Gas chromatograph-mass spectrometer (Agilent Technologies, model: 7890B/5977B GC-MS)

  8. DB-5ms GC Column (length 30 m, id 0.25 mm, film thickness 0.25 μm) (Agilent Technologies, catalog number: 122-5532)

  9. Gel Pump (Savant Instruments, GP110)

  10. Speed vac Plus (Savant Instruments, SC110A)

  11. Centrifuge (Eppendorf, catalog number: 5424)

  12. Thermomixer (Eppendorf ThermoMixer C, 5382000015)

Software

  1. MassHunter Workstation Software (Agilent Version B.07.01)

  2. MS Quantitation software (Agilent Version B.07.01/Build 7.1.524.0)

  3. Python 2.6+ (http://www.pythonxy.com)

  4. IsoCor Software (http://metasys.insa-toulouse.fr/software/isocor)

Procedure

Figure 1 is an overview of the procedures in this protocol.



Figure 1. The flow chart for this protocol. Animals are injected with the 15N tracer, and metabolites are extracted to analyze for 15N-labeled metabolites using GC MS.



  1. Tracer Injection

    1. Freshly prepare 15NH4Cl in PBS (see Recipe 1).

    2. Weigh mice and calculate the volume for the injection.

    3. Intraperitoneal injection (IP) of 15NH4Cl at 167 mg/kg or the same volume of PBS.


  2. Tissue collection

    1. Mice were quickly sacrificed with cervical dislocation at different time points after injection. For example, 0 min, 5 min, 15 min, 30 min and 60 min.

      Note: Different metabolites can reach their peak enrichment at different time points depending on the tissue.

    2. Enucleate mouse eyes and isolate neural retina and the eyecups under dissecting microscope in cold HBSS. Store the neural retina in a pre-weighed 1.5 ml microtube and the eyecup in a pre-weighted 2 ml microtube. Weigh the tissues and snap-freeze in liquid nitrogen.

    3. Perform cardiac puncture to withdraw 100-300 µl blood using a syringe with a 26G needle. Gently dispense the blood to EDTA-containing tubes. Place the blood samples on ice. Centrifuge the blood at 1,008 × g for 10 min at 4 °C, transfer the supernatant into a new 1.5 ml tube, and store supernatant in -20 °C refrigerator.

    4. Quickly remove brain and liver tissue, store them in a pre-weighed 2 ml microtube and snap-freeze them in liquid nitrogen.

      Note: Take out the whole brain to avoid heterogeneity from different regions. Cut a small piece of liver tissue (~40 mg) from the same lobe with scissors.


  3. Metabolite Extraction

    1. Tissue metabolite extraction (Figure 2)



      Figure 2. A schematic for metabolite extraction and derivatization. A. The brain, liver and eyecups were homogenized with an Omni THb Homogenizer, while the retina was homogenized with a pestle motor mixer. The homogenates were left on dry ice for 30 min and then centrifuged at 25,200 × g at 4 °C for 15 min. The supernatant was transferred to a glass insert containing 5 µl internal standard and dried with a speed vacuum. B. Add 10 µl of freshly prepared methoxyamine (20 mg/ml) into the dried samples in the insert and incubate for 90 min at 37 °C in a thermomixer, followed by a 30 min incubation at 70 °C after the addition of 30 µl of TBDMS. Transfer each insert into vials for GC MS analysis.


      1. Pre-chill extraction buffer (Recipe 2) on dry ice for 10 min.

      2. Transfer pre-chilled extraction buffer into tubes with tissues. Homogenize neural retina with a handheld pestle motor mixer in 140 µl extraction buffer for 15-20 s; Homogenize eyecup with an Omni THb Homogenizer in 200 µl extraction buffer for 15-20 s; Homogenize brain or liver tissues with the Omni THb Homogenizer for 20-30 s in extraction buffer (add 200 µl extraction buffer for every 5 mg tissues).

        Note: Change the disposable pestle for each sample. Clean the probe of Omni Thb homogenizer at least twice with clean water and wipes between samples to avoid cross-contamination. Leave the samples on dry ice for 30 min.

      3. Centrifuge the samples at 25,200 × g at 4 °C for 15 min.

      4. Filter the supernatants with a 0.2 µm syringe filter.

      5. Add 5 µl internal standard (Recipe 3) to each glass insert in 1.5 ml tube.

      6. Transfer the supernatant to each glass insert, open the tube lid to dry in a Speed vac in the cold room.

        Note: To ensure high sensitivity without overloading, transfer all the supernatant from retina or eyecup samples to dry and transfer 50 µl of brain or liver tissues to dry. Make sure the samples were fully dried. The moisture can affect the efficiency in derivatization and ionization.

    2. Plasma metabolite extraction

      1. Mix 10 µl plasma sample with 40 µl pre-chilled extraction buffer.

      2. Leave the mixture on ice for 15 min.

      3. Centrifuge the samples and transfer 10 µl into inserts with internal standard as described in tissue metabolite extraction.


  4. Sample preparation (Figure2)

    1. Freshly prepare methoxyamine mix (Recipe 4) and add 10 µl to each insert with the dried sample inside a 1.5 ml microtube. Mix and close the lid, then incubate the tube at 37 °C and 300 RPM for 90 min in a thermomixer.

      Note: Gently tap the tubes with inserts inside 3-5 times to mix, spin the samples down and close the lid tightly.

    2. After incubation, quickly spin the tubes and add 30 µl TBDMS to each sample. Incubate at 70 °C for 30 min.

    3. Spin down the tubes and transfer each insert into GC MS glass vials.


  5. GC-MS Analysis

    1. Pre-run preparation

      1. Install GC MS with DB-5 MS column and syringe needle.

      2. Set up a flow rate of helium gas at 1 ml/min.

      3. Fill the needle washing solvent vials A and B with hexane and methylene chloride respectively.

      4. Set up the GC oven temperature gradient as Table 1. The total run for each sample takes 31.5 min.


        Table 1. The parameters for GC oven temperature


      5. Set up the parameters for GC MS scan range, speed, frequency, cycle time and step size as Table 2. Choose the solvent delay for 5.4 min.


        Table 2. The parameters for GC MS scan range and speeder parameters on GC-MS



      6. Set up parameters for selection monitoring (SIM) mode as Table 3 for the ions of each metabolite that will be monitored.


        Table 3. List of ions for metabolites that are monitored under SIM mode


        Note: Isotopologuesare named as M0, M1, M2. M0 is the mass without labeling, and 1 to 2 represents the mass shift from the isotope labeling.


      7. Set up injection volume as 1 µl of the sample in split-less mode.

      8. Save these parameters as “GC MS nitrogen method”.

      9. Tune GC MS with autotune to check mass accuracy and ensure no leakage in the system.

      Note: Air leakage and dirty source in the system can significantly decrease the sensitivity. It is critical to tune the system weekly with regular replacement of the air trap and source maintenance.

    2. Run sample

      1. Place samples in the auto-sampler.

      2. Fill the sequence table for sample names and vial names. Select GC MS nitrogen method.

      3. Run samples with GC MS.

        Note: Check the instrument that it runs properly, especially for the long run. The typical instrument running failure includes an improperly filled sequence table, defective syringe needle and malfunctional filament.

    3. GC MS data processing

      1. Set up a quantitation method using Agilent MS Quantitation software based on Table 3.

      2. Click “File” in the software to set up a new batch.

      3. Select the quantitation method and extract the peak area for each selected ion.

      4. Export the peak area into an excel file.


  6. Nature abundance correction

    1. Nature abundance correction

      1. Install IsoCor for natural abundance correction software

        Download IsoCor software at http://metasys.insa-toulouse.fr/software/isocor; Download Python 2.6+ (http://www.pythonxy.com) and install modules: wxPython (v 2.8.11.0), NumPy( v1.6.0.2), SciPy( v0.9.0.1).

      2. Set up parameters for IsoCor software

      3. Edit “Metabolites. dat” file. Open the “Metabolites.dat” file under the IsoCor folder with Notepad and input metabolites and their elemental formula in Table 4. Save the file in the same folder.


        Table 4. List of metabolites and their elemental formula for natural abundance correction



      4. Edit “Derivatives. dat” file. Using Notepad to edit this file and input the chemical derivatives with their elemental formulas as Table 5. Set up the input data file as Table 6 and copy the GC MS peak area (ion intensity) into this table. Save the file in the same folder.


        Table 5. List of chemical derivatives with their elemental formula


        TBDMS1, 2 or 3 represents the number of TBDMS in the derivative metabolite.


    2. Data correction

      1. Save the Excel Input data file (Table 6) as “xxx. txt.”

      2. Open IsoCor software and select Isotopic tracer as “N”.

      3. Select the purity of the tracer as (0.02; 0.98).

        Note: The purity is dependent on the tracer you are using. For example, the purity of 15NH4Cl is 98%. It is represented as (0.02; 0.98).

      4. Load the input data file using the “Load multiple means” button.

      5. Click on the “Process” button.

      6. Open a new Excel file and load Input data file_res.txt.

        Note: The output of the calculations is automatically saved in a .txt file as (Input File_res.txt) and (Input File_log.txt). Save them in the same folder.Table 7 is a representative output file after natural abundance correction.


        Table 6. Template of Input data file for IsoCor

        The intensity is representative data from the extracted peak area for specifically monitored ion.

Data analysis

Representative data



Figure 3. GC MS chromatogram from standards and mouse tissue samples. A. GC MS chromatogram for M0 glutamate (m/z 432.3) from an amino acid standard mix. The calibration curve for M0 glutamate was calculated using the standard mix. B. The chromatogram of M0 glutamate (m/z 432.3) was extracted from the TIC in mouse retina sample received PBS injection.


Figure 3 is the representative GC MS chromatogram from standards and mouse tissule samples. Table 7 is the representative GC MS data that after natural abundance correction from liver tissue after injection with 15NH4Cl. Isotopologue distribution is the enrichment of 15N from the tracer. Except for branch chain amino acids including leucine, isoleucine, and valine, all the other metabolites reach their peak enrichment at 5 min after single tracer injection. The enrichment drops at 15 min due to metabolic degradation.


Table 7. The representative GC MS data of isotopologue distribution of liver tissue at 5 min and 15 min after natural abundance correction Isotopogue distribution

Recipes

  1. 15NH4Cl- solution

    Weigh ammonium-15N Chloride and dissolve in filtered 1× PBS at 33 mg/ml

  2. Extraction buffer

    Mix methanol and HPLC water at 80:20 (Vol:Vol)

  3. Internal Standard

    Weigh myristic acid-D27 powder and dissolve in Isopropanol: methanol: HPLC water mixture at 2:5:2 ratio (Vol:Vol:Vol)

  4. Methoxyamine mix

    Dissolve methoxyamine hydrochloride in pyridine solution at 20 mg/ml

    Note: Take the pyridine solution with a syringe needle through the sealed rubber lid to avoid moisture.

Acknowledgments

National Institutes of Health Grant EY026030 and EY031324 (To JD), BrightFocus Foundation (To JD), and the Retina Research Foundation (To JD) supported this work.

Competing interests

The authors declare no conflicts of interest.

Ethics

Mouse experiments were performed in accordance with the National Institutes of Health guidelines and the protocol (#1611004455, 01/18/2020-01/17/2023) was approved by the Institutional Animal Care and Use Committee of West Virginia University.

References

  1. Albertino, B., Borgialli, R., Guerra, M. G., Gasparri, G., Oliaro, A. and Dei Poli, M. (1989). Sequential study of the immunologic picture in neoplasm patients before and after surgical intervention. Minerva Chir 44(17): 1917-1920.
  2. Chokkathukalam, A., Kim, D. H., Barrett, M. P., Breitling, R. and Creek, D. J. (2014). Stable isotope-labeling studies in metabolomics: new insights into structure and dynamics of metabolic networks. Bioanalysis 6(4): 511-524.
  3. Du, J., Linton, J. D. and Hurley, J. B. (2015). Probing Metabolism in the Intact Retina Using Stable Isotope Tracers. Methods Enzymol 561: 149-170.
  4. Du, J., Yanagida, A., Knight, K., Engel, A. L., Vo, A. H., Jankowski, C., Sadilek, M., Tran, V. T., Manson, M. A., Ramakrishnan, A., Hurley, J. B. and Chao, J. R. (2016). Reductive carboxylation is a major metabolic pathway in the retinal pigment epithelium. Proc Natl Acad Sci U S A 113(51): 14710-14715.
  5. Fiehn, O. (2002). Metabolomics--the link between genotypes and phenotypes. Plant Mol Biol 48(1-2): 155-71.
  6. Fiehn, O. (2016). Metabolomics by Gas Chromatography-Mass Spectrometry: Combined Targeted and Untargeted Profiling. Curr Protoc Mol Biol 114: 30-34 31-30 34 32.
  7. Grenell, A., Wang, Y., Yam, M., Swarup, A., Dilan, T. L., Hauer, A., Linton, J. D., Philp, N. J., Gregor, E., Zhu, S., Shi, Q., Murphy, J., Guan, T., Lohner, D., Kolandaivelu, S., Ramamurthy, V., Goldberg, A. F. X., Hurley, J. B. and Du, J. (2019). Loss of MPC1 reprograms retinal metabolism to impair visual function. Proc Natl Acad Sci U S A 116(9): 3530-3535.
  8. Jang, C., Chen, L. and Rabinowitz, J. D. (2018). Metabolomics and Isotope Tracing. Cell 173(4): 822-837.
  9. Wang, Y., Bai, C., Ruan, Y., Liu, M., Chu, Q., Qiu, L., Yang, C. and Li, B. (2019). Coordinative metabolism of glutamine carbon and nitrogen in proliferating cancer cells under hypoxia. Nat Commun 10(1): 201.
  10. Xu, R., Ritz, B. K., Wang, Y., Huang, J., Zhao, C., Gong, K., Liu, X. and Du, J. (2020). The retina and retinal pigment epithelium differ in nitrogen metabolism and are metabolically connected. J Biol Chem 295(8): 2324-2335.
  11. Yam, M., Engel, A. L., Wang, Y., Zhu, S., Hauer, A., Zhang, R., Lohner, D., Huang, J., Dinterman, M., Zhao, C., Chao, J. R. and Du, J. (2019). Proline mediates metabolic communication between retinal pigment epithelial cells and the retina. J Biol Chem 294(26): 10278-10289.

简介

[摘要]含氮代谢物,包括氨,氨基酸和核苷酸,对于细胞代谢,生长和神经传递至关重要。在氨基酸和核苷酸的分解和生物合成中,氮代谢与碳代谢紧密相关。核磁共振光谱法和质谱法(包括气相色谱-质谱法(GC MS)和液相色谱法(LC MS))均已用于测量氮代谢。在这里,我们描述了使用15 N氨气与GC MS结合追踪多种小鼠组织中氮代谢的方案。该协议在 包括示踪剂注射,组织制备,代谢物提取,GC MS分析和自然丰度校正的详细程序。该协议将为研究代谢活性组织(例如视网膜,脑,肝和肿瘤)中组织特异性氮提供有用的工具。

[背景]核磁共振波谱法(NMR)和质谱法(包括质谱法(GC MS)和液相色谱法(LC MS))已成功用于各种生物体的全系统代谢物测量(Fiehn,2002和20 1 6; Chokkathukalam等,2014)。但是,每种方法都有其局限性,取决于研究类型,包括绝对定量,代谢物性质,灵敏度,稳健性,同位素分析和成本效益(Chokkathukalam等人,2014)。与NMR相比,基于MS的方法具有更高的灵敏度和代谢物覆盖率,因此更加常用。稳定的同位素标记加上LC MS或GC MS可以灵敏地定量健康和患病组织或细胞中的动态代谢变化(Jang等人,2018)。LC MS与稳定的氮同位素结合揭示了谷氨酰胺氮代谢如何与癌细胞中的碳代谢协调(Wang等人,2019)。GC MS具有出色的色谱分离度和成本效益(Jang等,2018)。在这里,我们开发了一种稳定的方法,可以使用稳定的氮同位素与GC MS结合定量氮衍生的代谢物。W¯¯ Ë确定了几个关键代谢奥利奇在视网膜设有该方法中,包括神经视网膜内和视网膜和视网膜色素上皮(RPE)之间的代谢通信(Albertino等人,1989;都。等人,2015年和2016 ; Grenell等,2019;Yam等,2019)。最近,我们使用15 N氨来追踪小鼠体内的氮代谢,并揭示了组织特异性代谢途径(Xu等人,2020年)。在此协议中,我们描述了在小鼠中进行氮示踪剂注射,组织制备,代谢物提取,GC MS样品制备,仪器分析和自然丰度校正的程序。

关键字:气质联用, 质谱分析法, 氮代谢, 15N示踪, 氨代谢, 氨基酸, 氨, 稳定同位素示踪剂

材料和试剂
1. 2毫升微管
2. 1.5毫升管     
3.注射器用仪26针(BD,CA talog号:9304308)     
4.注射器为0.2μ米注射器过滤器(BD,CA talog号:7212651)                   
5.用于GC进样的ALS注射器(Agilent Technologies,目录号:5181-3354)     
6.微型管(Axygen Scientific,目录号:02820037,1.5 ml,目录号:12018028,2 ml)     
7. 0.2μ米注射器过滤器(Thermo Scientific的,目录号:00293112)     
8. GC MS样品瓶盖(安捷伦科技公司,目录号:5182-0717)     
9.玻璃插件(Agilent Technologies,目录号:5181-2085,规格:250μl)     
10.一次性杵(Argos Technologies,目录号:7339-901 ) 
11. C57 B6 / J鼠标(杰克逊实验室,目录号:664) 
12.乙醇(Fisher Chemical,目录号:172382,LOT特定浓度:96%) 
13.液氮 
14.甲醇(Fisher Chemical,Optima TM LC / MS等级,目录号:164905) 
15. HPLC水(Fisher Chemical,Optima TM LC / MS级,目录号:7732-18-5) 
16.己烷(Sigma-Aldrich,HPLC级,目录号:MKCF5755) 
17.二氯甲烷(Fisher Chemical,目录号:163938) 
18.氦气(MATHESON,目录号:HE UHP1A) 
19.吡啶(Sigma-Aldrich,目录号:SHBK1583) 
20. N-叔丁基二甲基甲硅烷基-N-甲基三氟乙酰胺(TBDMS)(Sigma-Aldrich,目录号:394882) 
21.铵-15 N氯化物(15 NH 4 Cl)(Sigma-Aldrich,目录号:229251) 
22.氨基酸标准混合物(Sigma-Aldrich,目录号:AAS18-5 ml) 
23. 10 ×磷酸盐缓冲盐水(PBS)(Fisher Chemical,目录号:10010049) 
24.汉克平衡盐溶液(HBSS)(Fisher Chemical,目录号:14170112) 
25. EDTA(Fisher Chemical,目录号:6N011324) 
26. 15 NH 4 Cl溶液(参见配方) 
27.提取缓冲区(请参阅食谱) 
28.内部标准(请参见食谱) 
29.甲氧胺混合物(请参阅食谱) 


设备


Dumont Tweezer,样式5(电子显微镜科学,目录号:0108-5-PO)
-20°C冰箱
解剖显微镜(Zeiss,型号:Stemi 2000-C)
用于小鼠和大鼠的FirstHand外科手术器械套件(肯特科学公司,型号:INSSMOUSEKIT)
电池供电的杵式电动搅拌机(Argos Technologies,目录号:EW-44468-25 )
Omni组织匀浆器(115V 125W)(OMNI International the Homogenizer Company,THB-01)
气相色谱质谱仪(Agilent Technologies,型号:7890B / 5977B GC-MS)
DB-5ms GC色谱柱(长度30 m,内径0.25 mm,膜厚0.25μm)(Agilent Technologies,目录号:122-5532)
凝胶泵(Savant仪器(GP110)
Speed vac Plus(Savant Instruments,SC110A)
离心机(Eppendorf,货号:5424)
Thermomixer(Eppendorf ThermoMixer C,5382000015)

软件


MassHunter工作站软件(安捷伦B.07.01版)
MS定量软件(安捷伦版本B.07.01 /构建7.1.524。0 )
Python 2.6以上版本(http://www.pythonxy.com)
IsoCor软件(http://metasys.insa-toulouse.fr/software/isocor)

程序


图1是此协议中过程的概述。



图1 。该协议的流程图。向动物注射15 N示踪剂,并提取其代谢物,以使用GC MS分析15 N标记的代谢物。


示踪剂注入
新鲜编写,J重新15 NH 4氯在PBS(小号EE ř ecipe 1)。
称重小鼠并计算注射量。
腹腔注射(IP)15 NH 4 Cl,剂量为1 67 mg / kg或相同体积的PBS。

组织收集
在注射后的不同时间点,使小鼠迅速处死并使其脱臼。例如,0分钟,5分钟,15分钟,30分钟和60分钟。
注意:根据组织的不同,不同的代谢物可以在不同的时间点达到其峰富集。

在解剖型HBSS中,在解剖显微镜下去核小鼠眼睛并分离神经视网膜和眼杯。将神经视网膜存储在预先称重的1.5 ml微管中,将眼杯存储在预先称重的2 ml微管中。称量组织并在液氮中速冻。
使用带有26G针头的注射器进行心脏穿刺以抽取100-300μl血液。轻轻将血液分配到含EDTA的试管中。将血液样本放在冰上。离心血液在1 ,008 ×克在4 10分钟℃,将上清转移到新的1.5ml管中,并存储在上清液-20℃的冰箱中。
快速去除脑和肝组织,将它们存储在预先称重的2 ml微管中,并在液氮中速冻。
注意:请抽出整个大脑以避免不同区域的异质性。用剪刀从同一瓣切下一块肝组织(约40 mg)。


代谢物提取
组织代谢物提取(图2)

图2 。代谢物提取和衍生化的示意图。一。用Omni THb均质器将脑,肝和眼杯均质化,而将视网膜用研棒电动搅拌器均质化。将匀浆在干冰上放置30分钟,然后在25 °C ,200 × g下于4 °C离心15分钟。将上清液转移至含有5 µl内标物的玻璃插入物中,并用高速真空干燥。B.甲DD 10微升的F reshly p repared甲氧胺(20毫克/毫升)插入在插入并孵育90分钟的干燥的样品在37℃下在70在恒温,随后孵育30分钟℃,之后将加入30 µl TBDMS。将每个插入物转移到样品瓶中,以进行GC MS分析。


在干冰上将提取缓冲液(配方2)预冷10分钟。
将预冷的提取缓冲液转移到装有组织的试管中。在140 µl提取缓冲液中,使用手持式研杵电动搅拌器将神经视网膜匀浆15-20 s;用Omni THb均质器在200 µl提取缓冲液中均质化眼罩15-20 s;使用Omni THb均质器在提取缓冲液中均质化大脑或肝脏组织20-30 s (每5 mg组织添加200 µl提取缓冲液)。
注意:为每个样品更换一次性杵。用清水将Omni Thb均质机的探针至少清洗两次,并在样品之间擦拭以避免交叉污染。将样品放在干冰上30分钟。

离心机将样品在25 ,200 ×克于4 ℃下15分钟。
用0.2 µm注射器过滤器过滤上清液。
在1.5 ml试管中的每个玻璃插件中加入5 µl内标物(配方3)。
将上清液转移到每个玻璃插入物上,打开管盖,在冷藏室中的Speed vac中干燥。
不是:为确保高灵敏度而又不超载,请将视网膜或眼罩样品中的所有上清液转移至干燥处,并将50 µl脑或肝组织转移至干燥处。确保样品完全干燥。水分会影响衍生化和电离的效率。
血浆代谢物提取
将10 µl血浆样品与40 µl预冷的提取缓冲液混合。
将混合物在冰上放置15分钟。
离心样品,并按照组织代谢物提取中的说明,将10 µl转移至具有内标的插入物中。

样品制备(图2)
新鲜制备甲氧基胺混合物(配方4),向每个插入物中添加10 µl ,将干燥的样品放入1.5 ml微管中。混合并盖上盖子,然后在热混合器中将试管在37°C和300 RPM下孵育90分钟。
注意:轻轻敲打带有插入物的试管至内部3-5次以进行混合,向下旋转样品并盖紧盖子。

孵育后,快速旋转试管,并向每个样品中添加30 µl TBDMS。在70 °C下孵育30分钟。
旋转试管,并将每个插入物转移到GC MS玻璃小瓶中。

GC-MS分析
运行前准备
我nstall GC MS用DB-5 MS柱和注射器针头。
将氦气的流速设置为1 ml / min。
分别用己烷和二氯甲烷填充针洗溶剂瓶A和B。
按照表1设置GC柱箱温度梯度。每个样品的总运行时间为31.5分钟。

表1. GC柱箱温度参数


设置GC MS扫描范围,速度,频率,循环时间和步长的参数,如表2所示。选择5.4分钟的溶剂延迟。

表2. GC-MS上GC MS扫描范围的参数和加速器参数


如表3所示,为要监测的每种代谢物的离子设置用于选择监测(SIM)模式的参数。

表3.在SIM模式下监控的代谢物离子列表

注意:同位素共聚体分别命名为M0,M1,M2。M0是未标记的质量,并且1到2表示从同位素标记开始的质量偏移。


在不分流模式下将进样量设置为样品的1 µl。
将这些参数保存为“ GC MS氮气法”。
使用自动调谐功能对GC MS进行调谐,以检查质量准确性并确保系统中没有泄漏。
注意:系统中的漏气和脏污源会大大降低灵敏度。每周定期更换气阱并进行气源维护非常重要。

运行样本
将样品放在自动进样器中。
在序列表中填写样品名称和样品瓶名称。选择GC MS氮气法。
使用GC MS运行样品。
注意:检查仪器是否正常运行,尤其是长期运行。典型的仪器运行故障包括序列表填充不正确,注射器针头有缺陷以及灯丝故障。

GC MS数据处理
使用基于表3的安捷伦MS定量软件设置定量方法。
在软件中单击“文件”以设置新批次。
选择定量方法并提取每个选定离子的峰面积。
将峰面积导出到excel文件中。

自然丰度校正
自然丰度校正
安装IsoCor进行自然丰度校正软件
在http://metasys.insa-toulouse.fr/software/isocor下载IsoCor软件;下载Python 2.6+(http://www.pythonxy.com)并安装模块:wxPython(v 2.8.11.0),NumPy(v1.6.0.2),SciPy(v0.9.0.1)。

设置IsoCor软件的参数
E dit“代谢产物。dat”文件。使用记事本打开IsoCor文件夹下的“ Metabolites.dat”文件,并在表4中输入代谢物及其元素公式。将文件保存在同一文件夹中。

表4.用于自然丰度校正的代谢物列表及其元素公式


编辑“衍生工具。dat”文件。使用记事本编辑此文件并输入化学衍生物及其元素公式,如表5所示。将输入数据文件设置为表6,然后将GC MS峰面积(离子强度)复制到该表中。将文件保存在同一文件夹中。

表5.化学衍生物及其元素式列表

TBDMS1、2或3表示衍生物代谢物中的TBDMS数。


数据校正
将Excel Input数据文件(表6 )另存为“ xxx”。文本。”
打开IsoCor软件,然后将同位素示踪剂选择为“ N”。
选择示踪剂的纯度为(0.02; 0.98)。
注意:纯度取决于您使用的示踪剂。例如,15 NH 4 Cl的纯度为98%。它表示为(0.02; 0.98)。

使用“加载多种方式”按钮加载输入数据文件。
点击“处理”按钮。
打开一个新的Excel文件并加载输入数据file_res.txt。
注意:计算结果将自动保存为.txt文件,分别为(Input File_res.txt)和(Input File_log.txt)。将它们保存在同一文件夹中。表7是代表了看跌自然丰度修正后的文件。





表6 。IsoCor的输入数据文件模板

强度是从提取的峰面积中得到的代表数据,用于特定监控的离子。

数据分析


代表数据



图3 。标准品和小鼠组织样品的GC MS色谱图。A.氨基酸标准混合物中谷氨酸M0的GC MS色谱图(m / z 432.3)。使用标准混合物计算谷氨酸M0的校准曲线。B.接受PBS注射的小鼠视网膜样品中TIC的谷氨酸M0(m / z 432.3)色谱图。

请在文本中引用图3 (引用)。


图3是标准品和小鼠组织样品的代表性GC MS色谱图。表7是代表性的GC MS数据,该数据是在注射15 NH 4 Cl后从肝脏组织中自然富集校正后得出的。同位素同位素分布是示踪剂中15 N的富集。除包括亮氨酸,异亮氨酸和缬氨酸的支链氨基酸外,所有其他代谢物在一次示踪剂注射后5分钟达到其峰富集。由于代谢降解,富集在15分钟时下降。





表7.自然丰度校正后5分钟和15分钟时肝组织的同位素同位素分布的代表性GC MS数据


配方小号


15 NH 4氯离子搜索解决方案Ñ
称重15 N氯化铵,并以33 mg / ml的浓度溶于已过滤的1 × PBS中

提取缓冲液
以80:20(Vol:Vol)混合甲醇和HPLC水

内部标准
称取肉豆蔻酸-D27粉末并以2:5:2的比例(Vol:Vol:Vol)溶于异丙醇:甲醇:HPLC水混合物

甲氧胺混合物
迪斯olve methox yamine盐酸盐在吡啶中于20毫克/毫升溶液

注意:用注射器针头将吡啶溶液穿过密封的橡胶盖,以防潮湿。


致谢


健康格兰特EY026030全国学院和EY031324 (公司JD),Brigh TF OCUS基金会(公司JD)和视网膜研究基金会(公司JD)支持这项工作。


利益争夺


作者宣称没有利益冲突。


伦理


根据美国国立卫生研究院的指导方针进行了小鼠实验,并且实验方案(#1611004455,01/18 / 2020-01 / 17/2023)得到了西弗吉尼亚大学的机构动物护理和使用委员会的批准。


参考


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Du J.,Linton,JD和Hurley,JB(2015)。使用稳定的同位素示踪剂探测完整视网膜中的代谢。方法酶561:149-170。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Xu, R., Wang, Y. and Du, J. (2021). Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry . Bio-protocol 11(4): e3925. DOI: 10.21769/BioProtoc.3925.
  2. Xu, R., Ritz, B. K., Wang, Y., Huang, J., Zhao, C., Gong, K., Liu, X. and Du, J. (2020). The retina and retinal pigment epithelium differ in nitrogen metabolism and are metabolically connected. J Biol Chem 295(8): 2324-2335.
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