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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

RX Rong Xu
YW Yekai Wang
JD Jianhai Du

发布: 2021年02月20日第11卷第4期 DOI: 10.21769/BioProtoc.3925 浏览次数: 4827

评审: Ramu KakumanuRohit JainAmit Kumar Dey

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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) search Mass spectrometry (质谱分析法) search Nitrogen metabolism (氮代谢) search 15N tracing (15N示踪) search Ammonia metabolism (氨代谢) search Amino acids (氨基酸) search Ammonia (氨) search Stable isotope tracer (稳定同位素示踪剂) search

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: 00 293112 )

  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

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

生物化学 > 其它化合物

神经科学 > 基础技术

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