A Gas Chromatography-Mass Spectrometry-Based Two Stage Assay for Measurement of in vitro myo-Inositol 3-phosphate Synthase (INO1) Activity

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Molecular Microbiology
Feb 2014



This method describes an in vitro assay for measuring INO1 enzyme activity (the conversion of glucose 6-phosphate to myo-inositol 3-phosphate) in cell-free extracts. The method was first described for Plasmodium falciparum cells in MacRae et al. (2014) and consists of two parts: Part 1 describes the assay itself while part 2 describes analysis of the myo-inositol 3-phosphate product using gas chromatography-mass spectrometry (GC-MS).

Materials and Reagents

  1. Cells to be assayed [in the development of this protocol, we used Plasmodium falciparum (3D7 strain) cell cultures and human red blood cells (kindly supplied by the Australian Red Cross)]
  2. 13C-U-glucose (Cambridge Isotope Laboratories, catalog number: CLM1396 )
    Note: 13C-U-glucose refers to Universally-labelled glucose - i.e. where all six carbons are 13C-atoms.
  3. Hexokinase (>130 Units/mg) (Sigma-Aldrich, catalog number: H4502 )
  4. Adenosine triphosphate (ATP) (Sigma-Aldrich, catalog number: A6419 )
  5. Magnesium chloride (MgCl2, AnalaR) (VWR International, catalog number: 25108.260 )
  6. Tris-HCl (pH 7.5) (Sigma-Aldrich, catalog number: T5941 )
  7. Ammonium chloride (NH4Cl) (Ajax, catalog number: 31-500G )
  8. Nicotinamide adenine dinucleotide (NAD+) (Sigma-Aldrich, catalog number: N3014 )
  9. NaHEPES (pH 7.4) (Sigma-Aldrich, catalog number: H3375 )
  10. Ethylene glycol tetraacetic acid (Sigma-Aldrich, catalog number: E3889 )
  11. Dithiothreitol (Sigma-Aldrich, catalog number: D0362 )
  12. scyllo-Inositol (hereafter abbreviated to ‘sI’) (Sigma-Aldrich, catalog number: I8132 )
  13. Glucose 6-phosphate (Sigma-Aldrich, catalog number: G7879 )
  14. myo-Inositol 3-phosphate (Cayman Chemical Company, catalog number: CAY10007778 )
  15. Chloroform (HPLC grade) (Thermo Fisher Scientific, catalog number: 10615492 )
  16. Methanol (HPLC grade) (Thermo Fisher Scientific, catalog number: 10767665 )
  17. Assay buffer (see Recipes)
  18. Lysis buffer (see Recipes)

    Additional materials required for GC-MS
  19. Methoxyamine hydrochloride (Sigma-Aldrich, catalog number: 226904-25G )
  20. Pyridine (Sigma-Aldrich, catalog number: 270970 )
  21. BSTFA + 1% TMCS (Sigma-Aldrich, Supelco, catalog number: 33148 )
  22. Glucose (Sigma-Aldrich, catalog number: G8270 )


  1. 1.5 ml tubes (with safe-lock lids) (Eppendorf)
  2. Bench-top centrifuge for 1.5 ml tubes
  3. 1 ml, 200 µl, and 20 µl pipettes and accompanying tips
  4. 37 °C water bath
  5. Boiling water bath
  6. Timer
  7. Distilled (e.g. MilliQ) water supply
  8. Light microscope, slides, and cover slips (for assessment of lysis)
  9. Bench-top vortex or water bath sonicator (may be required for efficient lysis)

    Additional equipment required for GC-MS
  10. Gas Chromatography-Mass Spectrometer (e.g. Agilent 7890B-5977A)
  11. DB-5MS + DG column, (30m x 0.25 mm, with 10 m inert gap) (Agilent, J&W)
  12. Ultra high purity helium
  13. 1.5 ml tubes (Eppendorf)
  14. 2 ml glass vials for mass spectrometry (e.g. Agilent, part number: 5182-0715 )
  15. 9 mm caps with septa for glass vials (e.g. Agilent, part number: 5185-5820 )
  16. 250 µl vial inserts (glass) (Agilent, part number: 5183-2085 )
  17. Rotary vacuum concentrator (e.g. Christ, model: RVC 2-33 CD Plus )
  18. Microdispensers and accompanying glass capillaries (50 µl adjustable, 200 µl) (Drummond Scientific Company)


  1. ChemStation software (MSD ChemStation D.01.02.16) (Agilent)


Note: An overview of the whole procedure can be seen in the accompanying figure.

Figure 1. Inositol 3-phosphate synthase assay: overview

  1. Generation of 13C-glucose 6-phosphate
    1. 13C-glucose 6-phosphate is generated by incubation of [300 µM]final 13C-U-glucose with [66 µg ml-1]final hexokinase in assay buffer at 37 °C for 10 min, followed by boiling for 10 min in order to denature the hexose kinase. The tubes are then transferred to ice.
      Note: We always produced the 13C-glucose 6-phosphate in bulk – enough for all reactions in the experiment. We reconstituted lyophilised hexokinase powder (>130 U/mg) in water at 10 mg/ml. This was added to the assay buffer at 6.8 per 1 ml assay buffer, to give a concentration of 68 μg/ml. A stock of 10 mM 13C-U-glucose was made (in water) and was added to the hexokinase/assay buffer at 30 μl per 1 ml, to give a final concentration of 300 μM 13C-U-glucose and 66 μg/ml hexokinase. Unused reaction products were stored at -80 °C for future use.
    2. Efficiency of this conversion is usually ~100% and can be assessed by standard gas chromatography-mass spectrometry (GC-MS) protocols for sugar/sugar-phosphate analysis [see below, MacRae et al. (2014) and Saunders et al. (2011) for details].

  2. Generation of cell lysates and INO1 assay
    1. Lysates of your cells of choice are prepared by suspending washed cell pellets (with >10 pellet volumes of ice-cold PBS, two times) in ice-cold lysis buffer for 10 min.
      1. When measuring INO1 activity in Plasmodium falciparum-infected red blood cells, cells were suspended at a concentration of 109 cells per ml buffer (as determined by cell counting at the point of the second PBS wash). However, the volume of the suspension buffer may need to be varied depending on total cell number available and in vivo activity of INO1.
      2. The lysate should be checked (by light microscopy) to confirm successful lysis, some agitation (by either brief vortexing or sonication may be required).
    2. Aliquots of 13C-glucose 6-phosphate substrate (50 µl) and cell lysate (50 µl) are combined, vortex mixed briefly (<1 sec), incubated in a water bath at 37 °C, and the reaction stopped at required time-points by boiling for 5 min. The tubes are then transferred to ice.
      Note: In our experiments, we found that maximal labelling was achieved after ~3 h, although we also included later time points. A typical experiment would include 0, 0.5, 1, 5, 10, 30, 60, 120, 180, and 240 min, and an additional ‘overnight’ time point, if required.
    3. Reaction solutions are then centrifuged at 16,100 x g and 4 °C for 5 min (to pellet cellular debris) and the supernatant (containing reaction products) transferred to a fresh 1.5 ml tube.
    4. Polar products are recovered by solvent extraction of 50 µl of the boiled assay mixture, following addition of chloroform (50 µl), methanol (150 µl), and MilliQ water (100 µl, containing 1 nmol sI as an internal standard), resulting in a final solvent ratio of chloroform/methanol/water (1:3:3 v/v/v). After vigorous vortex mixing (30 sec) and centrifugation at 16,100 x g and 4 °C for 5 min, this mixture results in two phases, a lower chloroform phase (~50 µl) and an upper methanol/water phase (~300 µl).
    5. Analysis of 13C-glucose 6-phosphate and the synthesised 13C-myo-inositol 3-phosphate can be easily quantified by GC-MS, by comparison to authentic standards, as described below and in MacRae et al. (2014) and Saunders et al. (2011).

  3. Sample preparation for gas chromatography-mass spectrometry (GC-MS)
    1. An aliquot of the upper polar phase (100 µl) is transferred to a GC-MS vial insert (placed in a 1.5 ml microfuge tube) and dried in a rotary vacuum concentrator (RVC). Once dry, an additional 100 µl of the polar phase is added to the insert and dried. This process is repeated until almost all of the upper phase has been transferred and dried.
      Note: Be careful not to transfer any of the lipid-rich lower phase or the protein-rich interphase - it is advised to leave a few µl of the polar phase in the portioning tube rather than risk accidental transfer of the interphase and lower phase. Any minor differences in volume transferred are corrected later by normalisation to the internal standard (sI).
    2. To ensure all water is displaced from the dried polar phase, 40 µl of methanol is added and dried in the RVC, followed by an additional 40 µl methanol and subsequent drying.
    3. The vial inserts are transferred to 2 ml glass vials with forceps, being careful not to touch the rim of the insert.
    4. Polar metabolites are methoximated by addition of a freshly prepared solution of 20 mg/ml methoxyamine in pyridine (20 µl, this should be prepared when required, i. e. while the second aliquot of methanol is being dried).
    5. The vials are capped, vortexed briefly (~5 sec) and incubated at RT for >16 h. The caps are removed and methoximated samples derivatised by addition of 20 µl of BSTFA + 1% TMCS, a reagent that results in trimethylsilyl (TMS) substitutions on the relevant metabolites, allowing them to be observable by GC-MS. The vials are re-capped, vortexed briefly and incubated at RT for >1 h before injection onto the GC-MS.

  4. Gas chromatography-mass spectrometry (GC-MS)
    1. Derivatised samples are analyzed using a DB-5MS + DG column in splitless mode (injection temperature 270 °C), or equivalent, using ultra high purity helium as the carrier gas.
    2. The initial oven temperature is 70 °C (2 min), followed by temperature gradients to 295 °C at 12.5 °C/min, and from 295 °C to 320 °C at 25 °C/min. The final temperature is held for 3 min.
    3. Data analysis can be performed using brand-specific specific or non-brand-specific software (e.g. AnalyzerPro, Amdis, mzMatch, mzMine, etc.). We used the accompanying ChemStation software.
    4. Metabolites are identified by comparison of retention times and ion fragmentation patterns with authentic standards. Quantification of metabolites is calculated using the formula: amount metabolite (nmol) = (area of appropriate metabolite peaks/area of sI peak) x (1 nmol sI/metaboliteMRRFsI); where metaboliteMRRFsI is the molar relative response factor determined from the mean value of: area of appropriate metabolite peak/area of sI peak (1:1 standards).
      1. The ‘appropriate peaks’ are the extracted ion chromatogram (EIC) peaks of appropriate unlabelled and labelled ions for each metabolite (see below).
      2. The level of labelling is estimated as the percent of the metabolite pool containing one or more 13C atoms, after background subtraction for naturally occurring isotopes (as calculated from an unlabelled standard), as described in Zamboni et al. (2009).
    5. Ions used for quantification and label incorporation calculations:
      scyllo-Inositol (sI, internal standard): M0 = m/z 318
      myo-Inositol 3-phosphate: M0-M4 = m/z 318-322
      Glucose: M0-M4 = m/z 319-323
      Glucose 6-phosphate: M0-M2 = m/z 357-359
      Note: M0 represents the unlabelled ion (where all carbon is 12C), Mn represents ions with n 13C-atoms.


  1. Assay buffer
    1 mM ATP
    2.5 mM MgCl2
    100 mM Tris.HCl (pH 7.5)
    14 mM NH4Cl
    0.8 mM NAD+
  2. Lysis buffer
    1 mM NaHEPES (pH 7.4)
    2 mM ethylene glycol tetraacetic acid
    2 mM dithiothreitol


This work was supported by a project grants from the National Health and Medical Research Council of Australia (NHMRC). M.J.M is an NHMRC Principal Research Fellow and J.I.M. was supported by a Royal Society Travelling Fellowship.


  1. MacRae, J. I., Lopaticki, S., Maier, A. G., Rupasinghe, T., Nahid, A., Cowman, A. F. and McConville, M. J. (2014). Plasmodium falciparum is dependent on de novo myo‐inositol biosynthesis for assembly of GPI glycolipids and infectivity. Mol Microbiol 91(4): 762-776.
  2. Saunders, E. C., Ng, W. W., Chambers, J. M., Ng, M., Naderer, T., Kromer, J. O., Likic, V. A. and McConville, M. J. (2011). Isotopomer profiling of Leishmania mexicana promastigotes reveals important roles for succinate fermentation and aspartate uptake in tricarboxylic acid cycle (TCA) anaplerosis, glutamate synthesis, and growth. J Biol Chem 286(31): 27706-27717.
  3. Zamboni, N., Fendt, S. M., Ruhl, M. and Sauer, U. (2009). (13)C-based metabolic flux analysis. Nat Protoc 4(6): 878-892.


该方法描述了用于测量无细胞提取物中INO1酶活性(葡萄糖-6-磷酸转化为肌醇-3-磷酸)的体外测定。 该方法首先在MacRae等人(2014)中描述了恶性疟原虫细胞,并由两部分组成:第1部分描述测定本身,而第2部分描述分析 使用气相色谱 - 质谱(GC-MS)测定肌醇3-磷酸产物。


  1. 要测定的细胞[在本方案的开发中,我们使用恶性疟原虫(3D7株)细胞培养物和人红细胞(由澳大利亚红十字会友情提供)]
  2. < sup> 13 C-U-葡萄糖(Cambridge Isotope Laboratories,目录号:CLM1396)
    注意: 13 C-U-葡萄糖是指通用标记的葡萄糖,即其中所有六个碳都是 13个C原子。
  3. 己糖激酶(> 130单位/mg)(Sigma-Aldrich,目录号:H4502)
  4. 腺苷三磷酸(ATP)(Sigma-Aldrich,目录号:A6419)
  5. 氯化镁(MgCl 2,AnalaR)(VWR International,目录号:25108.260)
  6. Tris-HCl(pH7.5)(Sigma-Aldrich,目录号:T5941)
  7. 氯化铵(NH 4 Cl)(Ajax,目录号:31-500G)
  8. 烟酰胺腺嘌呤二核苷酸(NAD +)(Sigma-Aldrich,目录号:N3014)
  9. NaHEPES(pH7.4)(Sigma-Aldrich,目录号:H3375)
  10. 乙二醇四乙酸(Sigma-Aldrich,目录号:E3889)
  11. 二硫苏糖醇(Sigma-Aldrich,目录号:D0362)
  12. 肌动蛋白(以下缩写为'em'I')(Sigma-Aldrich,目录号:I8132)。
  13. 葡萄糖-6-磷酸(Sigma-Aldrich,目录号:G7879)
  14. 肌醇3-磷酸(Cayman Chemical Company,目录号:CAY10007778)
  15. 氯仿(HPLC级)(Thermo Fisher Scientific,目录号:10615492)
  16. 甲醇(HPLC级)(Thermo Fisher Scientific,目录号:10767665)
  17. 测试缓冲区(参见配方)
  18. 裂解缓冲液(见配方)

    GC-MS 所需的其他材料
  19. 甲氧基胺盐酸盐(Sigma-Aldrich,目录号:226904-25G)
  20. 吡啶(Sigma-Aldrich,目录号:270970)
  21. BSTFA + 1%TMCS(Sigma-Aldrich,Supelco,目录号:33148)
  22. 葡萄糖(Sigma-Aldrich,目录号:G8270)


  1. 1.5 ml管(带安全锁盖)(Eppendorf)
  2. 台式离心机用于1.5 ml管
  3. 1 ml,200μl和20μl移液器和附带的提示
  4. 37°C水浴
  5. 沸水浴
  6. 计时器
  7. 蒸馏(例如 MilliQ)供水
  8. 光学显微镜,载玻片和盖玻片(用于评价裂解)
  9. 台式涡流或水浴超声仪(可能需要有效裂解)

    GC-MS 所需的其他设备
  10. 气相色谱 - 质谱仪(例如Agilent 7890B-5977A)
  11. DB-5MS + DG柱(30m×0.25mm,具有10m惰性间隙)(Agilent,J& W)
  12. 超高纯氦
  13. 1.5ml管(Eppendorf)
  14. 用于质谱的2ml玻璃小瓶(例如Agilent,部件号:5182-0715)
  15. 带玻璃小瓶隔片的9 mm帽(例如Agilent,部件号:5185-5820)
  16. 250μl小瓶插入物(玻璃)(Agilent,部件号:5183-2085)
  17. 旋转真空浓缩器(例如 Christ,型号:RVC 2-33 CD Plus)
  18. 微分配器和伴随的玻璃毛细管(50μl可调,200μl)(Drummond Scientific Company)


  1. 化学工作站软件(MSD ChemStation D.01.02.16)(Agilent)




  1. 产生13 C-葡萄糖-6-磷酸
    1. 通过将[300μM]终末期13号CU-葡萄糖与[66μg/ml]葡萄糖一起温育,产生13 C-葡萄糖-6-磷酸。 -1]终止己糖激酶在测定缓冲液中37℃  10分钟,然后煮沸10分钟以变性 己糖激酶。然后将试管转移到冰上 注意: 我们 总是产生 13 C-葡萄糖6-磷酸盐 反应。我们重组冻干己糖激酶 粉末(> 130U/mg)的10mg/ml水溶液。将其加入到测定中 缓冲液,每1ml测定缓冲液为6.8,得到68μg/ml的浓度。制备(在水中)10mM的α-1,3-葡萄糖的储备液,并加入 以每1ml30μl的己糖激酶/测定缓冲液,得到最终的 浓度的300μM的13 C-U-葡萄糖和66μg/ml的己糖激酶。没用过 反应产物储存在-80℃以备将来使用。
    2. 这种转化的效率通常为〜100%并且可以通过评价 标准气相色谱 - 质谱(GC-MS)方案 糖/糖 - 磷酸分析[见下文,MacRae等人(2014)和 Saunders等人(2011)了解详情]。

  2. 生成细胞裂解物和INO1测定
    1. 选择的细胞的裂解物通过悬浮洗涤的细胞制备 丸粒(具有> 10个丸粒体积的冰冷PBS,两次) 冰冷的裂解缓冲液中10分钟。
      1. 测量时 INO1活性在恶性疟原虫感染的红细胞,细胞中 以10 9个细胞/ml缓冲液的浓度悬浮(as 通过在第二次PBS洗涤时的细胞计数确定)。 然而,悬浮液缓冲液的体积可能需要改变 取决于INO1的总细胞数和体内活性。
      2. 应检查裂解物(通过光学显微镜检查)以确认 成功裂解,一些搅拌(通过短暂涡旋或 可能需要超声处理)。
    2. 等分的13 C-葡萄糖-6-磷酸 底物(50μl)和细胞裂解物(50μl)混合,涡旋混合 短暂(<1秒),在37℃的水浴中温育, 反应在所需时间点通过煮沸5分钟停止。 管   然后转移到冰 注意: 在我们的实验中,我们 发现最大标记在〜3 h后实现,虽然我们也 包括以后的时间点。典型的实验将包括0,0.5, 1分钟,5分钟,10分钟,30分钟,60分钟,120分钟,180分钟和240分钟, 时间点,如果需要。
    3. 然后离心反应溶液 在16,100×g和4℃下5分钟(使细胞碎片沉淀),并且 上清液(含有反应产物)转移到新鲜的1.5ml  管。
    4. 极性产物通过50的溶剂萃取回收 加入氯仿(50μl) μl),甲醇(150μl)和MilliQ水(100μl,含有1nmol In 作为内标),导致最终溶剂比为 氯仿/甲醇/水(1:3:3v/v/v)。剧烈涡旋混合后 (30秒)并在16,100×g和4℃下离心5分钟,这 混合物产生两相,较低的氯仿相(〜50μl)和 上层甲醇/水相(约300μl)
    5. 13-葡萄糖的分析 6-磷酸和合成的肌醇3-磷酸可以是 通过GC-MS容易地定量,通过与真实标准比较 描述于下文和MacRae等人 (2014)和Saunders 等人(2011)。

  3. 气相色谱 - 质谱(GC-MS)的样品制备
    1. 将上极性相的等分试样(100μl)转移至GC-MS 小瓶插入物(置于1.5ml微量离心管中)并在旋转器中干燥 真空浓缩器(RVC)。 一旦干燥,额外100微升的极性 相加入到插入物中并干燥。 重复该过程直到 几乎所有的上层相都被转移并干燥 注意: 小心不要转移任何富含脂质的下相或 富含蛋白质的相间 - 建议离开几μl的极性 阶段在分配管中而不是风险意外转移   相间和下相。 转移的任何小的差异   通过归一化为内标(sI)进行校正。
    2. 为了确保所有的水从干燥的极性相中移出,40μl 的甲醇并在RVC中干燥,然后再另外40℃ μl甲醇,然后干燥
    3. 将小瓶插入物转移到具有镊子的2ml玻璃小瓶中,小心不要接触插入物的边缘。
    4. 极性代谢物通过加入新鲜制备物而肟化   20mg/ml甲氧基胺的吡啶溶液(20μl,这应该是 在需要时准备,i。 e。 而第二等份的甲醇是 干燥)。
    5. 盖上小瓶,短暂涡旋(约5秒) 并在室温下温育> 16小时。 除去帽并甲羟肟酸 样品通过加入20μlBSTFA + 1%TMCS(试剂)衍生化 其导致在相关的三甲基甲硅烷基(TMS)取代 代谢物,使其可以通过GC-MS观察到。 小瓶是 重新盖帽,短暂涡旋并在RT孵育≥1小时 注射到GC-MS上。

  4. 气相色谱 - 质谱(GC-MS)
    1. 衍生的样品使用不分流的DB-5MS + DG柱分析   模式(注射温度270℃)或等效,使用超高 纯度氦作为载气。
    2. 初始炉温 为70℃(2分钟),然后在12.5℃下温度梯度至295℃ ℃,以25℃/min从295℃至320℃。 最终温度为   保持3分钟。
    3. 可以使用进行数据分析 品牌特定的特定或非特定品牌的软件(例如 AnalyzerPro,Amdis,mzMatch,mzMine,等)。 我们用随附的 ChemStation软件。
    4. 通过比较鉴定代谢物 的保留时间和离子碎片模式与真实 标准。 代谢物的定量使用 公式:量代谢物(nmol)=(适当代谢物的面积 峰值/峰值/峰值面积)×(1nmol/l/m)代谢物/MRRF值)。 其中代谢物是由以下确定的摩尔相对反应因子: 平均值:适当代谢物峰的面积/峰的面积 (1:1标准)。
      1. "适当的峰"是 提取离子色谱图(EIC)峰的适当的未标记和 每个代谢物的标记离子(见下文)。
      2. 的级别 标记估计为含有代谢物库的百分比 一个或多个 13 C原子 (从未标记的标准计算),as 描述于Zamboni et al。 (2009)。
    5. 用于定量和标记结合计算的离子:
      scyllo -Inositol( s I,internal standard):M 0 = m/ z 318
      肌醇3-磷酸:M sub -M sub 4 = m/em z 318-322
      葡萄糖:M M4 = m/ 319-323
      葡萄糖6-磷酸:M sub 0 -M sub 2 = m/z 357-359
      em] 。


  1. 测定缓冲区
    1 mM ATP
    2.5mM MgCl 2 v/v 100mM Tris-盐酸(pH7.5) 14mM NH 4 Cl
    0.8 mM NAD +
  2. 裂解缓冲液
    1mM NaHEPES(pH7.4) 2mM乙二醇四乙酸 2mM二硫苏糖醇


这项工作得到澳大利亚国家卫生和医学研究委员会(NHMRC)的项目资助。 M.J.M是NHMRC首席研究员和J.I.M. 得到了皇家学会旅行奖学金的支持。


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  2. Saunders,E.C.,Ng,W.W.,​​Chambers,J.M.,Ng,M.,Naderer,T.,Kromer,J.O.,Likic,V.A.and McConville,M.J。(2011)。 利什曼原虫墨西哥前鞭毛体的同位素体分析揭示了琥珀酸发酵和天冬氨酸在三羧酸循环中的吸收的重要作用(TCA )anaplerosis,glutamate synthesis,and growth。 J Biol Chem 286(31):27706-27717。
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引用:MacRae, J. I. and McConville, M. J. (2015). A Gas Chromatography-Mass Spectrometry-Based Two Stage Assay for Measurement of in vitro myo-Inositol 3-phosphate Synthase (INO1) Activity. Bio-protocol 5(5): e1418. DOI: 10.21769/BioProtoc.1418.