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Quantification of 2-Hydroxyglutarate Enantiomers by Liquid Chromatography-mass Spectrometry
液相色谱质谱法量化2-羟戊二酸对映体   

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Abstract

Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al., 1980; Kranendijk et al., 2012). More recently, investigators have shown that D2HG is produced by mutant isocitrate dehydrogenase enzymes associated with a variety of human malignancies, such as acute myeloid leukemia, glioblastoma multiforme, and cholangiocarcinoma (Cairns and Mak, 2013; Dang et al., 2009; Ward et al., 2010). By contrast, we and others have shown that L2HG accumulates in response to cellular reductive stressors like hypoxia, activation of hypoxia inducible factors, and mitochondrial electron transport chain defects (Oldham et al., 2015; Reinecke et al., 2011; Intlekofer et al., 2015; Mullen et al., 2015). Each enantiomer is produced and metabolized in independent biochemical pathways in reactions catalyzed by separate enzymes and utilizing different cofactors with presumably different consequences for cellular metabolism (Kranendijk et al., 2012). Therefore, as research into the roles of D2HG and L2HG in human metabolism continues, it becomes increasingly important for investigators to consider each enantiomer independently (Struys, 2013). Several methods for quantification of biochemically relevant enantiomers in general have been developed and typically include enzymatic assays using enzymes specific for one enantiomeric species or the other, the use of chiral chromatography medium to facilitate chromatographic separation of enantiomers prior to spectroscopy, or the use of chiral derivatization reagents to convert a mixture of enantiomers to diastereomers with differing physical and chemical properties facilitating their chromatographic separation. In this protocol, we report the adaptation of a previously published derivatization method using diacetyl-L-tartaric anhydride (DATAN) for the quantification of 2HG enantiomers (Figure 1) (Oldham et al., 2015; Struys et al., 2004).


Figure 1. Reaction scheme for the derivatization protocol

Keywords: 2-hydroxyglutarate(2-羟基戊二酸二乙酯), Diacetyl-L-tartaric anhydride(diacetyl-l-tartaric酸酐), Liquid chromatography-mass spectrometry(液相色谱-质谱法), Hydrophilic liquid interaction chromatography(亲水-液相色谱)

Materials and Reagents

  1. Screw cap 2 ml microcentrifuge tubes (Fisher Scientific, catalog number: 21-403-200 )
  2. D-2-hydroxyglutaric acid disodium salt (Sigma-Aldrich, catalog number: H8378 )
  3. L-2-hydroxyglutaric acid disodium salt (Sigma-Aldrich, catalog number: 90790 )
  4. Sodium lactate (Sigma-Aldrich, catalog number: L7022 )
  5. Water (LC-MS grade) (Thermo Fisher Scientific, catalog number: W6 )
  6. [13C4]-2-oxoglutaric acid disodium salt (Cambridge Isotope Labs, catalog number: CLM-4442 )
  7. Methanol, Optima (LC-MS grade) (Thermo Fisher Scientific, catalog number: A454 )
  8. Diacetyl-L-tartaric anhydride (DATAN) (Sigma-Aldrich, catalog number: 358924 )
  9. Acetonitrile, Optima (LC-MS grade) (Fisher Scientific, catalog number: A955 )
  10. Acetic Acid, Glacial (Fisher Scientific, catalog number: BP2401 )
  11. Formic Acid, Optima (LC-MS grade) (Fisher Scientific, catalog number: A117 )
  12. Ammonium Hydroxide (Fisher Scientific, catalog number: A669 )
  13. Biological samples (see Note 1)
  14. 50 mM D2HG and L2HG stock solution (see Recipes)
  15. 20 μM 2HG working solution (see Recipes)
  16. 10 mM sodium lactate stock solution (see Recipes)
  17. 500 μM internal standard (ISTD) stock solution (see Recipes)
  18. Buffer A (see Recipes)
  19. Buffer B (see Recipes)

Equipment

  1. Savant SpeedVac ISS110
    Note: This device has been discontinued by Thermo Fisher Scientific.
  2. Isotemp digital dry bath incubator (Fisher Scientific, catalog number: 11-715-125DQ )
  3. Sequant ZIC-HILIC HPLC column (3.5 μm, 100 Å, 2.1 mm internal diameter, 150 mm length) (Merck Millipore Corporation, catalog number: 1504420001 )
  4. Sequant ZIC-HILIC HPLC guard column (Merck Millipore Corporation, catalog number: 1504360001 )
  5. Surveyor autosampler plus
    Note: This device has been discontinued by Thermo Fisher Scientific.
  6. Surveyor MS Pump Plus 
    Note: This device has been discontinued by Thermo Fisher Scientific.
  7. Finnigan LTQ mass spectrometer
    Note: This device has been discontinued by Thermo Fisher Scientific.

Software

  1. Xcalibur software (Thermo Fisher Scientific)

Procedure

  1. Preparation of standards
    1. Standards are prepared in 80% methanol by combining 0-20 μl of the racemic 20 μM 2HG working solution with 2 μl each of the 10 mM sodium lactate and 500 μM ISTD stocks into screw cap microcentrifuge tubes (Table 1). Lactate facilitates derivatization of the low concentration of 2HG found in the standards.
    2. Standards are vortexed and briefly centrifuged.

      Table 1. Preparation of 2HG standards


  2. Derivatization
    1. Metabolite extracts (200 μl) are transferred to 2 ml screw cap microcentrifuge tubes.
    2. Standards and biological samples are evaporated to dryness using a Speedvac roto-evaporator with drying rate set to “HIGH” (~65 °C oven temperature). Complete evaporation of all water is critically important as the derivatization reaction will not occur in the presence of water. Evaporation may also be performed under nitrogen gas (Note 2).
    3. Derivatization reagent is prepared by dissolving DATAN in acetonitrile:acetic acid (4:1, v/v) to 50 mg/ml. The esterification reaction requires an aprotic solvent. Acetonitrile is more polar than dimethyl chloride, and we found this solvent more capable of dissolving the dried residue. Additionally, acetonitrile was directly compatible with our chromatographic approach.
    4. Dried samples are treated with 50 μl DATAN solution and heated at 70 °C for 2 h in a temperature controlled heat block. The derivatized standard solutions change color from clear to clear-yellow, while samples change from clear to dark yellow-brown. We previously used derivatization times as short as 30 min, but have found that 2 h results in consistently complete derivatization.
    5. Samples are cooled to room temperature and spun briefly.
    6. Derivatized samples are diluted with 50 μl acetonitrile:acetic acid (4:1, v/v), vortexed, spun briefly, and transferred to a microcentrifuge tube for LC-MS analysis.
      Note: Diluting in acetonitrile:acetic acid was critically important for achieving adequate separation of the enantiomers chromatographically. Importantly, this method does not require an additional evaporation and resuspension step prior to LC-MS analysis as required by other published protocols. 

  3. Liquid chromatography
    1. Separation is performed on a ZIC-HILIC stationary phase with guard column.
    2. Mobile phases are prepared from 200 mM formic acid in LC-MS grade water titrated to pH 3.25 with ammonium hydroxide, acetonitrile, and LC-MS grade water.
    3. The autosampler wash solution is 95% LC-MS grade acetonitrile in LC-MS grade water.
    4. Samples are injected at 5 μl using a 20 μl sample injection loop.
    5. Liquid chromatography is performed using a 15% buffer A and 85% buffer B isocratic elution of 30 min per sample.

  4. Mass spectrometry
    1. The mass spectrometer is tuned using a standard solution of racemic 2HG to optimize ionization and ion optics.
    2. The mass spectrometer is operated in selective reaction monitoring mode, and the following mass transitions are monitored over the duration of the chromatographic run: 363 > 147 + 129 m/z (derivatized 2HG), 147 > 129 m/z (2HG), and 149 > 105 m/z (ISTD). We found the signal intensity for the 147 > 129 m/z transition was much greater than the 363 > 147 m/z transition and used the former for quantification of 2HG enantiomers. We confirmed that underivatized and derivatized 2HG peaks were well separated chromatographically, and confirmed the retention times of the derivatized samples using the 363 > 147 m/z transition in standard samples (Figure 2).


      Figure 2. Derivatization of 2HG standards. Standard solutions of D2HG (5 μM), L2HG (5 μM), and D2HG plus L2HG (5 μM each) were prepared at 5 μM in 200 μl of 80% methanol. These samples were evaporated, derivatized, and analyzed by LC-MS as described above. The 147 > 129 m/z extracted ion chromatogram of the racemic 2HG mixture (purple trace) demonstrated two well resolved peaks corresponding to the retention times observed for the derivatized D2HG sample (green trace) and the derivatized L2HG sample (blue trace). The green and blue traces are offset on the Y-axis by 5,000 intensity units for clarity. The 363 > 147 m/z extracted ion chromatogram (gray trace plotted on the right Y-axis) confirms these peaks represent derivatized 2HG. Underivatized 2HG elutes with a retention time of 6 min (not shown).

  5. Data analysis
    1. Chromatographic peak areas for D2HG, L2HG, and the ISTD are integrated using Xcalibur software.
    2. Peak area ratios are calculated (D2HG/ISTD and L2HG/ISTD) for the standards to generate a standard curve (Figure 3).
    3. The sample concentration for each 2HG enantiomer is interpolated from the standard curve.
    4. Sample concentrations of 2HG should be normalized to some measure of sample mass (cell count, cell protein, tissue mass).


      Figure 3. Representative standard curves

Representative data



Figure 4. A representative chromatogram from derivatized cellular extracts. Lung fibroblasts were treated with siRNA to silence expression of L2HGDH, the only enzyme known to metabolize L2HG. After 48 h, metabolites were extracted on dry ice using 80% methanol (Note 1). A 200 μl sample was evaporated and derivatized as described above. Peak areas were integrated using Xcalibur software and found to be: D2HG 967 arbitrary units (AU), L2HG 7,811 AU, and ISTD 65,470 AU (chromatogram not shown). Thus, the peak area ratios are: D2HG 0.015 and L2HG 0.119. The interpolated sample concentrations from the standard curve are: D2HG 51 nM and L2HG 348 nM. The mass of 2HG in the derivatized sample is determined by multiplying the concentration by the total volume, 100 μl. Therefore, the original sample contained 5.1 pmol D2HG and 34.8 pmol L2HG. We typically divide metabolite mass by the cell count for normalization. 

Notes

  1. Biological samples from cells, tissue, plasma, and urine can be prepared using a variety of methods (Yuan et al., 2012). For our studies of cultured cells in 6-well dishes, we wash once with 2 ml of ice cold phosphate buffered saline per well and transfer the plates to a bed of dry ice. We then add 1 ml of 80% aqueous methanol pre-cooled to -80 °C and 10 μl of ISTD. The plates are then incubated at -80 °C for 15 min. The cells are then collected by scraping into a microcentrifuge tube. Insoluble material is pelleted by centrifugation at 15,000 x g at 4 °C. The supernatant is transferred to a new tube and aliquoted for LC-MS analysis.
  2. We found the roto-evaporator more useful as it concentrates the residue into a small pellet at the bottom of the tube while the nitrogen tended to disperse the dried residue throughout the tube.
  3. An alternative derivatization reagent, TSPC, has recently been reported to have improved sensitivity compared to DATAN derivatization (Cheng et al., 2015).

Recipes

  1. 50 mM D2HG and L2HG stock solution
    Prepare 50 mM D2HG and L2HG stock solution in water
    Aliquot and store at -20 °C until use
  2. 20 μM 2HG working solution
    1. Dilute the 50 mM stocks to 1 mM
      2 μl D2HG
      2 μl L2HG
      96 μl LC-MS grade water
    2. Further dilute 1 mM 2HG to 20 μM in LC-MS grade water
  3. 10 mM sodium lactate stock solution
    Prepare 10 mM in water
    Aliquot and store at -20 °C until use
  4. 500 μM internal standard (ISTD) stock solution
    Prepare 500 μM [13C4]-2-oxoglutaric acid disodium salt stock solution in methanol
    Store at -80 °C for use as an ISTD
    Note: One may also chemically reduce (e.g., with zinc) isotopically labeled 2-oxoglutarate prior to derivatization for an isotopically labeled racemic 2HG internal standard.
  5. Buffer A
    10% 200 mM formic acid
    90% LC-MS grade water
  6. Buffer B
    10% 200 mM formic acid
    90% acetonitrile

Acknowledgments

This protocol was adapted from a prior publication (Struys, 2013). The work was supported by the NIH/NHLBI (HL007633 to WMO, and HL061795, HL108630, and GM107618 to JL) and the Brigham and Women’s Hospital Department of Medicine.

References

  1. Chalmers, R. A., Lawson, A. M., Watts, R. W., Tavill, A. S., Kamerling, J. P., Hey, E. and Ogilvie, D. (1980). D-2-hydroxyglutaric aciduria: case report and biochemical studies. J Inherit Metab Dis 3(1): 11-15.
  2. Cheng, Q. Y., Xiong, J., Huang, W., Ma, Q., Ci, W., Feng, Y. Q. and Yuan, B. F. (2015). Sensitive Determination of onco-metabolites of D- and L-2-hydroxyglutarate enantiomers by chiral derivatization combined with liquid chromatography/mass spectrometry analysis. Sci Rep 5: 15217.
  3. Cairns, R. A. and Mak, T. W. (2013). Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Discov 3(7): 730-741.
  4. Duran, M., Kamerling, J. P., Bakker, H. D., van Gennip, A. H. and Wadman, S. K. (1980). L-2-Hydroxyglutaric aciduria: an inborn error of metabolism? J Inherit Metab Dis 3(4): 109-112.
  5. Dang, L., White, D. W., Gross, S., Bennett, B. D., Bittinger, M. A., Driggers, E. M., Fantin, V. R., Jang, H. G., Jin, S., Keenan, M. C., Marks, K. M., Prins, R. M., Ward, P. S., Yen, K. E., Liau, L. M., Rabinowitz, J. D., Cantley, L. C., Thompson, C. B., Vander Heiden, M. G. and Su, S. M. (2009). Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 462(7274): 739-744.
  6. Intlekofer, A. M., Dematteo, R. G., Venneti, S., Finley, L. W., Lu, C., Judkins, A. R., Rustenburg, A. S., Grinaway, P. B., Chodera, J. D., Cross, J. R. and Thompson, C. B. (2015). Hypoxia iduces production of L-2-hydroxyglutarate. Cell Metab 22(2): 304-311.
  7. Kranendijk, M., Struys, E. A., Salomons, G. S., Van der Knaap, M. S. and Jakobs, C. (2012). Progress in understanding 2-hydroxyglutaric acidurias. J Inherit Metab Dis 35(4): 571-587.
  8. Mullen, A. R., Hu, Z., Shi, X., Jiang, L., Boroughs, L. K., Kovacs, Z., Boriack, R., Rakheja, D., Sullivan, L. B., Linehan, W. M., Chandel, N. S. and DeBerardinis, R. J. (2014). Oxidation of alpha-ketoglutarate is required for reductive carboxylation in cancer cells with mitochondrial defects. Cell Rep 7(5): 1679-1690.
  9. Oldham, W. M., Clish, C. B., Yang, Y. and Loscalzo, J. (2015). Hypoxia-mediated increases in L-2-hydroxyglutarate coordinate the metabolic response to reductive stress. Cell Metab 22(2): 291-303.
  10. Reinecke, C. J., Koekemoer, G., van der Westhuizen, F. H., Louw, R., Lindeque, J. Z., Mienie, L. J., Smuts, I. (2012). Metabolomics of urinary organic acids in respiratory chain deficiencies in children. Metabolomics 8(2): 264-283.
  11. Struys, E. A. (2013). 2-Hydroxyglutarate is not a metabolite; D-2-hydroxyglutarate and L-2-hydroxyglutarate are!. Proc Natl Acad Sci U S A110(51): E4939.
  12. Struys, E. A., Jansen, E. E., Verhoeven, N. M. and Jakobs, C. (2004). Measurement of urinary D- and L-2-hydroxyglutarate enantiomers by stable-isotope-dilution liquid chromatography-tandem mass spectrometry after derivatization with diacetyl-L-tartaric anhydride. Clin Chem 50(8): 1391-1395.
  13. Ward, P. S., Patel, J., Wise, D. R., Abdel-Wahab, O., Bennett, B. D., Coller, H. A., Cross, J. R., Fantin, V. R., Hedvat, C. V., Perl, A. E., Rabinowitz, J. D., Carroll, M., Su, S. M., Sharp, K. A., Levine, R. L. and Thompson, C. B. (2010). The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 17(3): 225-234.
  14. Yuan, M., Breitkopf, S. B., Yang, X. and Asara, J. M. (2012). A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc 7(5): 872-881.

简介

2-羟基戊二酸(2HG),L(L2HG)和D(D2HG)的两种对映异构体是哺乳动物细胞中未知功能的代谢物,其最初与分开的和罕见的代谢的先天性错误相关,导致与神经系统相关的2HG的尿排泄增加儿童中的缺陷(Chalmers等人,1980; Duran等人,1980; Kranendijk等人,2012)。最近,研究者已经表明,D2HG由与多种人类恶性肿瘤相关的突变体异柠檬酸脱氢酶产生,所述人类恶性肿瘤例如急性骨髓性白血病,多形性成胶质细胞瘤和胆管癌(Cairns和Mak,2013; Dang等人,2009; Ward等人,2010)。相比之下,我们和其他人已经显示L2HG响应于细胞还原应激物如缺氧,缺氧诱导因子的激活和线粒体电子传递链缺陷而累积(Oldham等人,2015; Reinecke 2011; Intlekofer等人,2015; Mullen等人,2015年)。每种对映异构体在独立的生物化学途径中在由单独的酶催化的反应中产生和代谢,并且利用不同的辅因子,对细胞代谢可能具有不同的后果(Kranendijk等人,2012)。因此,随着对人类代谢中D2HG和L2HG的​​作用的研究不断进行,研究人员独立地考虑每种对映体变得越来越重要(Struys,2013)。已经开发了用于定量生物化学相关对映异构体的几种方法,并且通常包括使用对一种对映异构物种或另一种具有特异性的酶的酶测定,使用手性色谱介质以促进光谱之前对映异构体的色谱分离,或使用手性衍生化试剂将对映异构体的混合物转化为具有不同物理和化学性质的非对映异构体,促进它们的色谱分离。在该方案中,我们报告了使用二乙酰基-L-酒石酸酐(DATAN)用于定量2HG对映体的先前公开的衍生化方法的改编(图1)(Oldham等人,2015; Struys 。,2004)。



图1.衍生化方案的反应方案

关键字:2-羟基戊二酸二乙酯, diacetyl-l-tartaric酸酐, 液相色谱-质谱法, 亲水-液相色谱

材料和试剂

  1. 螺旋盖2ml微量离心管(Fisher Scientific,目录号:21-403-200)
  2. D-2-羟基戊二酸二钠盐(Sigma-Aldrich,目录号:H8378)
  3. L-2-羟基戊二酸二钠盐(Sigma-Aldrich,目录号:90790)
  4. 乳酸钠(Sigma-Aldrich,目录号:L7022)
  5. 水(LC-MS级)(Thermo Fisher Scientific,目录号:W6)
  6. [剑桥同位素实验室,目录号:CLM-4442]
  7. 甲醇,Optima(LC-MS级)(Thermo Fisher Scientific,目录号:A454)
  8. 二乙酰基-L-酒石酸酐(DATAN)(Sigma-Aldrich,目录号:358924)
  9. 乙腈,Optima(LC-MS级)(Fisher Scientific,目录号:A955)
  10. 乙酸,冰川(Fisher Scientific,目录号:BP2401)
  11. 甲酸,Optima(LC-MS级)(Fisher Scientific,目录号:A117)
  12. 氢氧化铵(Fisher Scientific,目录号:A669)
  13. 生物样品(见注1)
  14. 50 mM D2HG和L2HG储备溶液(见配方)
  15. 20μM2HG工作溶液(参见配方)
  16. 10 mM乳酸钠储备液(见配方)
  17. 500μM内标(ISTD)储备溶液(见配方)
  18. 缓冲区A(参见配方)
  19. 缓冲液B(参见配方)

设备

  1. Savant SpeedVac ISS110
    注意:此设备已被Thermo Fisher Scientific停止销售。
  2. Isotemp数字干浴培养箱(Fisher Scientific,目录号:11-715-125DQ)
  3. Sequencer ZIC-HILIC HPLC柱(3.5μm,内径2.1mm,长度150mm)(Merck Millipore Corporation,目录号:1504420001)
  4. Sequant ZIC-HILIC HPLC保护柱(Merck Millipore Corporation,目录号:1504360001)
  5. Surveyor自动进样器加上
    注意:此设备已被Thermo Fisher Scientific停止销售。
  6. Surveyor MS泵Plus 
  7. Finnigan LTQ质谱仪 注意:此设备已被Thermo Fisher Scientific停止使用。

软件

  1. Xcalibur软件(Thermo Fisher Scientific)

程序

  1. 准备标准
    1. 通过将0-20μl外消旋20μM2HG工作溶液与2μl各10mM乳酸钠和500μMISTD储备液混合到螺旋盖微量离心管中(表1),在80%甲醇中制备标准品。 乳酸盐促进标准品中发现的低浓度2HG的衍生化。
    2. 将标准品涡旋并短暂离心
      表1. 2HG标准品的制备


  2. 衍生化
    1. 将代谢物提取物(200μl)转移到2ml螺旋盖微量离心管中
    2. 使用Speedvac旋转蒸发器将标准品和生物样品蒸发至干燥,干燥速率设定为"高"(约65℃烘箱温度)。 所有水的完全蒸发是非常重要的,因为衍生化反应不会在水的存在下发生。 蒸发也可以在氮气下进行(注2)。
    3. 衍生试剂通过将DATAN溶于乙腈:乙酸(4:1,v/v)至50mg/ml制备。 酯化反应需要非质子溶剂。 乙腈的极性大于二甲基 氯化物,我们发现这种溶剂更能溶解干燥的残余物。此外,乙腈与我们的色谱方法直接兼容。
    4. 干燥的样品用50μlDATAN溶液处理,并在温度受控的加热块中在70℃加热2小时。衍生标准溶液的颜色从透明变为透明黄色,而样品从透明变为深黄棕色。我们以前使用的衍生化时间短至30分钟,但已经发现2小时导致一致的完全衍生化。
    5. 将样品冷却至室温并短暂旋转。
    6. 衍生化的样品用50μl乙腈:乙酸(4:1,v/v)稀释,涡旋,短暂旋转,并转移到微量离心管用于LC-MS分析。
      注意:在乙腈:乙酸中稀释对于通过色谱法实现对映体的充分分离是至关重要的。重要的是,该方法不需要在LC-MS分析前进行额外的蒸发和重悬步骤,如其他已公布的方案所要求。

  3. 液相色谱
    1. 在具有保护柱的ZIC-HILIC固定相上进行分离
    2. 流动相由用氢氧化铵,乙腈和LC-MS级水滴定至pH 3.25的LC-MS级水中的200mM甲酸制备。
    3. 自动进样器洗涤溶液是LC-MS级水中的95%LC-MS级乙腈。
    4. 使用20μl样品注射环以5μl注射样品
    5. 使用15%缓冲液A和85%缓冲液B等度洗脱每个样品30分钟进行液相色谱法。

  4. 质谱法
    1. 使用外消旋2HG的标准溶液调谐质谱仪以优化电离和离子光学。
    2. 质谱仪在选择性反应监测模式下操作,并且在色谱运行的持续时间内监测以下质量转变:363> 147 + 129m/z(衍生的2HG),147> 129 m/z(2HG),149> 105 m/z(ISTD)。我们发现147> 129 m/z跃迁远大于363> 147 m/z跃迁,并使用前者定量2HG对映体。我们证实未衍生化和衍生化的2HG峰在色谱上被良好分离,并且使用363>标记物证实了衍生化样品的保留时间。标准样品中147 m/z跃迁(图2)

      图2:2HG标准品的衍生制备D2μM(5μM),L2HG(5μM)和D2HG + L2HG(各5μM)的标准溶液,浓度为5μM,溶于200μl80%甲醇。将这些样品蒸发,衍生化,并如上所述通过LC-MS分析。 147>外消旋2HG混合物(紫色痕量)的129m/z提取离子色谱图显示了对应于对衍生的D2HG样品(绿色曲线)和衍生的L2HG样品(蓝色曲线)观察到的保留时间的两个良好分辨的峰。为了清楚起见,绿色和蓝色迹线在Y轴上偏移5,000个强度单位。 363> 147m/z提取离子色谱图(灰色曲线绘制在右Y轴上)证实这些峰代表衍生的2HG。未衍生的2HG洗脱,保留时间为6分钟(未示出)
  5. 数据分析
    1. 使用Xcalibur软件对D2HG,L2HG和ISTD的色谱峰面积进行积分。
    2. 计算峰面积比(D2HG/ISTD和L2HG/ISTD),以生成标准曲线(图3)。
    3. 从标准曲线插入每个2HG对映异构体的样品浓度。
    4. 2HG的样品浓度应该归一化为样品质量(细胞计数,细胞蛋白质,组织质量)的一些量度

      图3.代表性标准曲线

代表数据



图4.来自衍生化细胞提取物的代表性色谱图。肺部成纤维细胞用siRNA处理以沉默L2HGDH的表达,L2HGDH是已知代谢L2HG的唯一酶。 48小时后,使用80%甲醇在干冰上提取代谢物(注1)。将200μl样品蒸发并如上所述进行衍生化。使用Xcalibur软件积分峰面积,发现为:D2HG 967任意单位(AU),L2HG 7,811 AU和ISTD 65,470 AU(色谱图未显示)。因此,峰面积比为:D2HG 0.015和L2HG 0.119。来自标准曲线的内插样品浓度为:D2HG 51nM和L2HG 348nM。通过将浓度乘以总体积100μl来确定衍生化样品中2HG的质量。因此,原始样品含有5.1pmol D2HG和34.8pmol L2HG。我们通常将代谢物质量除以细胞计数以进行归一化。

笔记

  1. 来自细胞,组织,血浆和尿液的生物样品可以使用多种方法制备(Yuan等人,2012)。对于我们在6孔培养皿中培养的细胞的研究,我们用2ml冰冷的磷酸盐缓冲盐水/孔洗涤一次,并将板转移到干冰床。然后加入1ml预冷至-80℃的80%甲醇水溶液 和10μl的ISTD。然后将平板在-80℃下温育15分钟。然后通过刮入微量离心管中收集细胞。通过在4℃以15,000×g离心沉淀不溶性物质。将上清液转移到新管中并等分用于LC-MS分析。
  2. 我们发现旋转蒸发器更有用,因为它将残余物集中在管底部的小颗粒中,而氮气倾向于将干燥的残余物分散在整个管中。
  3. 与DATAN衍生化相比,另一种衍生化试剂TSPC最近已被报道具有改善的灵敏度(Cheng等人,2015)。

食谱

  1. 50mM D2HG和L2HG储备液
    准备50 mM D2HG和L2HG储备液的水溶液
    等分并存储在-20°C,直到使用
  2. 20μM2HG工作溶液
    1. 将50mM原液稀释至1mM
      2μlD2HG
      2μlL2HG
      96μlLC-MS级水
    2. 进一步稀释1 mM 2HG至20μM的LC-MS级水
  3. 10mM乳酸钠储液
    准备10mM水溶液
    等分并存储在-20°C,直到使用
  4. 500μM内标(ISTD)储备液
    在甲醇中制备500μM的[13 C] 4 - [2-氧代戊二酸二钠盐]储备溶液。
    存储在-80°C用作ISTD
    注意:在同位素标记的外消旋2HG内标的衍生化之前,还可以化学还原(例如用锌)同位素标记的2-氧戊二酸。
  5. 缓冲区A
    10%200mM甲酸
    90%LC-MS级水
  6. 缓冲区B
    10%200mM甲酸
    90%乙腈

致谢

该协议改编自以前的出版物(Struys,2013)。这项工作得到了NIH/NHLBI(HL007633到WMO,HL061795,HL108630和GM107618到JL)和Brigham and Women's Hospital Department of Medicine的支持。

参考文献

  1. Chalmers,RA,Lawson,AM,Watts,RW,Tavill,AS,Kamerling,JP,Hey,E。和Ogilvie,D。(1980)。  D-2-羟基戊二酸尿症:病例报告和生化研究 J Inherit Metab Dis 3 (1):11-15。
  2. 在这种情况下,我们可以使用一个简单的例子来说明这个问题。(a) ://www.ncbi.nlm.nih.gov/pubmed/26458332"target ="_ blank">通过手性衍生化与液相色谱/质谱分析结合,敏感测定D-和L-2-羟基戊二酸对映异构体的代谢产物。 Sci Rep 5:15217.
  3. Cairns,RA和Mak,TW(2013)。  致癌性异柠檬酸脱氢酶突变:机制,模型和临床机会。癌症发现 3(7):730-741。
  4. Duran,M.,Kamerling,JP,Bakker,HD,van Gennip,AH和Wadman,SK(1980)。  癌症相关的IDH1突变产生2-羟基戊二酸。 Nature 462(7274 ):739-744。
  5. 在这种情况下,我们的研究结果表明,这些研究结果表明,这些研究结果可以帮助我们更好地理解这些研究结果。(Intlekofer,AM,Dematteo,RG,Venneti,S.,Finley,LW,Lu,C.,Judkins,AR,Rustenburg,AS,Grinaway,PB,Chodera,JD,Cross,JRand Thompson, ; 缺氧产生L-2-羟基戊二酸。 Cell Metab 22(2):304-311。
  6. Kranendijk,M.,Struys,EA,Salomons,GS,Van der Knaap,MS and Jakobs,C。(2012)。  了解2-羟基戊二酸尿症的进展 J Inherit Metab Dis 35(4):571-587。 >
  7. Mullen,AR,Hu,Z.,Shi,X.,Jiang,L.,Boroughs,LK,Kovacs,Z.,Boriack,R.,Rakheja,D.,Sullivan,LB,Linehan, DeBerardinis,RJ(2014)。  氧化α-酮戊二酸是具有线粒体缺陷的癌细胞中的还原性羧化所必需的。细胞代 7(5):1679-1690。
  8. Oldham,WM,Clish,CB,Yang,Y.和Loscalzo,J.(2015)。  缺氧介导的L-2-羟基戊二酸的增加调节对还原应激的代谢反应。细胞Metab 22(2):291-303。 br />
  9. Reinecke,CJ,Koekemoer,G.,van der Westhuizen,FH,Louw,R.,Lindeque,JZ,Mienie,LJ,Smuts,I.(2012)。  尿有机酸在儿童呼吸链缺乏症中的代谢组学 代谢组学 em> 8(2):264-283。
  10. Struys,E.A。(2013)。  2-羟基戊二酸不是代谢物; D-2-羟基戊二酸和L-2-羟基戊二酸是美国国家科学院院报110(51):E4939。
  11. Struys,EA,Jansen,EE,Verhoeven,NM和Jakobs,C。(2004)。 
  12. Ward,PS,Patel,J.,Wise,DR,Abdel-Wahab,O.,Bennett,BD,Coller,HA,Cross,JR,Fantin,VR,Hedvat,CV,Perl,AE,Rabinowitz,JD,Carroll, M.,Su,SM,Sharp,KA,Levine,RL和Thompson,CB(2010)。  用于体液,细胞和新鲜和固定组织的正/负离子交换,靶向质谱法的代谢组学平台。 Nat Protoc 7 (5):872-881。
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引用:Oldham, W. M. and Loscalzo, J. (2016). Quantification of 2-Hydroxyglutarate Enantiomers by Liquid Chromatography-mass Spectrometry. Bio-protocol 6(16): e1908. DOI: 10.21769/BioProtoc.1908.
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