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13C Kinetic Labeling and Extraction of Metabolites from Adherent Mammalian Cells
13C 动力标记和提取附着哺乳动物细胞的代谢产物

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Abstract

Fluctuations in metabolite levels in mammalian cells are the most direct form of readout of the cellular metabolic state. The current protocol describes a method for pulse labeling and subsequent isolation of metabolites from adherent mammalian cells. The isolated metabolites can be identified and quantified by mass-spectrometry, allowing for estimation of the rates of synthesis and removal of metabolites from the system being analyzed.

Materials and Reagents

  1. THP1 cells (from ATCC, catalog number: TIB 202 )
  2. Methanol (LC-MS Ultra CHROMASOLV) (Sigma-Aldrich, catalog number: 14262 )
  3. Water (LC-MS Ultra CHROMASOLV) (Sigma-Aldrich, catalog number: 14263 )
  4. Acetonitrile (LC-MS Ultra CHROMASOLV) (Sigma-Aldrich, catalog number: 14261 )
  5. Hexane (Sigma-Aldrich, catalog number: 296090 )
  6. HCl (Sigma-Aldrich, catalog number: 258148 )
  7. Trypan blue (Gibco, catalog number: 15250-061 )
  8. Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
  9. Pyridine (Sigma-Aldrich, catalog number: 270970 )
  10. Sulfur trioxide pyridine (Sigma-Aldrich, catalog number: S7556 )
  11. Barium acetate (Sigma-Aldrich, catalog number: 243671 )
  12. D-Glucose (13C6 99%) (Euriso-Top, catalog number: CLM-481 )
  13. RPMI culture media 1640 (Gibco, catalog number: 31800-014 )
  14. Glucose free RPMI media-1640 (Sigma-Aldrich, catalog number: R1383 )
  15. Dialyzed FBS (Hyclone, catalog number: SH30071.03 )
  16. Cholate buffer (see Recipes)
  17. Quenching mix (see Recipes)

Equipment

  1. Bright-line hemacytometer (Sigma-Aldrich, catalog number: Z359629 )
  2. Cell scraper
  3. Culture dish
    6 well plate (Nunc, catalog number: 140685 )
    Flask (T-175, catalog number: 178983 )
  4. Liquid nitrogen cylinder
  5. Nitrogen gas cylinder
  6. Water bath
  7. Refrigerators (-20, -80, 4 °C)
  8. Microcentrifuge (Eppendrof, model: 5418R )
  9. Mono spin C18 plugs (GL Sciences, catalog number: 5010-21700 )
  10. HPLC (Agilent, model: 1260 infinity ; Pump: Binary Pump VL, model: G1312C )
  11. HPLC column
    Amino column (Polaris 5 NH2 150 * 2.0 mm)
    C18 column [ZORBAX Eclipse Plus C18 (Narrow Bore 2.1 * 150 mm 5 μ)]
    Cyano column (Phenomenax Luna 150 * 2.0 mm 3 μ)
  12. Mass spectrometer (ABSciex, hybrid 4000 QTrap)
  13. Amber glass vials (Supelco, catalog number: 29117 )
  14. Glass vials (Borosil, catalog number: 9910 )
  15. Glass pasture pipette
  16. Sonicator (Branson, model: 1210 )

Procedure

  1. Metabolite labeling and extraction (Mehrotra et al., 2014)
    1. Culture cells in desired media. For the current protocol, THP1 cells were cultured in RPMI media, supplemented with 10% FCS at 37 °C, 5% CO2. Confluent cells were counted (using trypan blue) and seeded in phorbol myristate acetate (PMA) containing complete media for differentiation at a density of 0.8 million cell/media complete media. The final concentration of PMA used was 30 ng/ml complete media and differentiation was allowed for 48 h. If one wishes to perform kinetic labeling, seed multiple sets of cells, depending on time points to be covered. e.g. seed separate sets of cells to extract metabolites after -0, 1, 2, 5, 15, 30 and 60 min of label introduction. (The PMA differentiation step is for THP1 cells. Seeding for other adherent mammalian cells can be performed as is done routinely. The number of cells to be seeded for metabolite extraction to get good signals on the mass-spectroscope has to be optimized for each cell type. The signals obtained in our case were derived from 5 million cells, seeded in 3 wells of 6 well plate in a total of 6 ml PMA containing complete media.)
      For labeling of cells with 13C6 glucose (or any other label), replace the media in culture dishes with fresh RPMI media supplemented with 10% dialyzed fetal calf serum one hour before introduction of label (it is important to use dialyzed serum as un-dialyzed forms are rich in small molecules including glucose). At the time of labeling, remove media from the culture dish and perform a quick wash of cells with glucose free media (the entire step should not exceed 30 sec- using 1-2 ml of media). This step is crucial to remove unlabeled glucose media from the culture dish.
    2. Add 13C6-glucose containing RPMI media, supplemented with 10% dialyzed fetal calf serum to the cells for the required amounts of time, which in our case ranged from 0 min to 60 min as mentioned earlier. For upto 60 min of incubation, agitation is not required, however for longer incubation time, gentle agitation in breaks may become important. (Glucose concentration-2 mg labeled glucose added per ml of glucose free RPM media, supplemented with 10% FCS.) Make sure that the media temperature is 37 °C since temperature fluctuations can induce changes in the metabolic profile.
    3. At the completion of incubation time, immediately remove the labeled media completely and add the quenching mix- chilled (-70 °C), methanol-water (80: 20) to the cells. For our study we added 700 µl of the quenching mixture which was sufficient to cover the surface of seeded cells. There is no need to wash cells with Glucose free media before adding the quenching mix as this will only increase the exposure time of cells to labeled media (further, since we did not measure glucose on the mass spectrometer, the left over quantity of label carrying 13C glucose did not make any difference to our observations). The quenching mix may be spiked with an external standard to gauge for any losses during extraction. (Example of an of external standard is Fumaric acid-13C4, d4. which shows a peak shift corresponding to M+8 from Fumaric acid. The standard can be added to the quenching mix (80:20 methanol: water) before using it for metabolic quenching. The concentration of the standard needs to be optimized and may be used in a range from 0.1 to 100 ng/ul of quenching mix).
    4. Immediately place the culture dishes in -75 °C for 10 min to allow for complete metabolic quenching. This is followed by incubation on ice for 10-15 min to allow for freeze-thaw lysis of cells.
    5. The cells are then scraped off the culture dish on dry ice.
    6. Vortex for 10 min with 30 sec of votexing followed by 1 min incubaton on ice.
    7. Spin the lysate at 6,000 x g for 5 min at 4 °C.
    8. Collect supernatant and add a 200 µl of quenching mix to the pellet and vortex hard. Re-spin the tube and collect the supernatant. Repeat step.
    9. Pool the three supernatants obtained (steps A9-10).
    10. Dry the supernatants under a stream of N2 gas. 1,000 µl of extract will dry up in approximately 20 min.
    11. Re-suspend the dried extract in MS grade water (re-suspension volume is dependent on cell number and MS-sensitivity range. For 5 million THP1 cells we used 180 µl volume).
    12. Pass the samples once through the mono spin C18 columns to remove any particulate debris (this allows for greater longevity of the LC column).
    13. Proceed for LC-MS analysis and the samples may be further diluted with acetonitrile or other organic solvents depending on the LC column requirements. It is best to analyze the isolated metabolites on the mass spectroscope within 24 h of isolation. Extracted metabolites should be stored at -80 °C. The extracts, while processing, re-suspension or mass analysis should be maintained at 4 °C.

  2. Lipid (fatty acids and cholesterol) labeling and extraction
    1. Fatty acid and cholesterol labeling
      1. Seed and label the cells as described in step A14. For lipid labeling experiments in our study, cells were incubated in the label carrying media for 4 h, unlike the labeling of metabolites, which was carried out for short intervals with maximum being 60 min. The cell number taken for lipid labeling experiments was 30 million, seeded in a T-175 flask.
      2. Completely remove the labeling media from the flask at the end of labeling time.
      3. Add cholate buffer to the flasks and incubate at RT for 5 min with gentle intermittent tapping. For 30 x 106 THP1 cells seeded in T-175 flasks, 3 ml of buffer was used. To collect cells efficiently, use a scraper.
      4. Collect the lysate and perform a hard vortex (vortex for 5 min, 30 sec vortex followed by 30 sec incubation on ice) followed by centrifugation at 3,000 x g for 10 min at 4 °C.
    2. Lipid extraction: Free fatty acid isolation
      Free fatty acids were obtained by following the protocol by Aveldano and Horrocks (1983) with slight modifications (Aveldano and Horrocks, 1983). For lipid extraction and handling, strictly glassware should be used. It is important to note here that nowhere during lipid extraction and handling should plastic ware be used.
      1. Extract lipids from the cells using the Bligh and Dyer protocol in Borosil glass tubes (Bligh and Dyer, 1959). Briefly, to the each 1 ml of lysate collected in step B1d; add 3.75 ml 1:2 CHCl3: MeOH and vortex well for a minute. Then add 1.25 ml of CHCl3 and vortex well. This is followed by the final addition of 1.25 ml of water and vortexing. The tubes are then centrifuged at 1,000 rpm for 5 min at room temperature to yield a two-phase system. Carefully extract the bottom layer as the lipid containing zone using a glass pasture pipette.
      2. Dry extracted lipids under a stream of nitrogen gas and store in -20 °C in N2 gas atmosphere until further use. Since lipids are extremely prone to peroxidation, store dried lipids in vials topped with nitrogen gas, in dark.
      3. Re-suspend the dried lipids in 100 μl of MS grade water and add 1 ml of 4:1 acetonitrile: 37% (v/v) hydrochloric acid.
      4. Cap the vials and incubate at 90 °C for 2 h to allow for acid hydrolysis of all triacyl glycerides, allowing for the release of fatty acids.
      5. Cool down the extracts to room temperature and add 1 ml of hexane, followed by vortexing for 20 sec.
      6. Leave the samples at RT for 5 min, undisturbed, followed by centrifugation at 3,000 x g for 5 min.
      7. Collect the supernatant, which is the layer with hydrolyzed lipids. Perform the same procedure of hexane addition to the pellet and pool the two supernatants.
      8. Measure the volume of collected sample accurately. Divide it into two equal halves and dry both sets under nitrogen stream.
      9. After drying the first set, add 200 µl of 50: 40: 5 chloroform: methanol: water and 0.01% aqueous ammonia, vortex well and use directly for Lipid analysis.
      10. Use the second set for cholesterol derivatization.
    3. Lipid extraction: Cholesterol derivatization
      Cholesterol molecule does not ionize well in the mass spectrometer. To aid the ionization process, cholesterol is converted to cholesterol sulphate by following the protocol by Sandhoff et al. (1999).
      1. In a fresh dry, glass amber vial, add 2.5 mg of sulfur trioxide pyridine, followed by addition of 2.5 mg of dry pyridine.
      2. Sonicate the contents for 10 sec in a bath sonicator using ultrasonics at room temperature..
      3. Add 20 µl of the solution from B3b to the vial containing the dried sample (i.e. to the sample stored at step B2j). The sample vial must be at RT. Sonicate in water bath for 10 sec.
      4. Leave the samples at RT for 15 min.
      5. At completion of incubation, add 2.1 μl of 314 mM solution of barium acetate. Sonicate for 10 sec in water bath.
      6. Incubate at RT for 10 min, followed by incubation at 4 °C for 60 min.
      7. At the completion of incubation, allow vials to come to RT. Add 120 µl of methanol and centrifuge the mix at 13,000 x g for 10 min.
      8. The supernatant can be directly used for mass spectrometric measurement of cholesterol.

  3. Results and interpretation
    As the labeled glucose moieties get metabolized in the cells, the quantity of down-stream metabolites carrying the label (formed by glucose catabolism) increases. In parallel, a decrease in their unlabeled isotopic form is observed. The phenomenon can be captured on the mass spectroscope.
    There are essentially two ways of data evaluation:
    1. Absolute metabolite quantitation and determination of formation/degradation rates
      1. Standard curves: Generate calibration curves on the mass spectrometer, for the metabolites to be monitored, by injecting standard solutions covering a range of concentrations.
      2. Determination of labeling transitions of metabolites: Analyze the mass spectrometry data obtained from the test samples, to carefully identify the labeling patterns in each of the metabolites. For example, when 13C6 Glucose is fed to THP1 cells, there are numerous possible labeling patterns (transitions) that can be obtained on the mass-spectrometer for Dihydroxy acetone phosphate (DHAP). However, if labeled glucose, catabolize via glycolysis, is the only major contributor to the molecule, then the most abundant transitions detected on the mass spectrometer will be either the completely unlabeled form or the one carrying all the labeled carbons.


        Figure 1. The two most abundant labeling patterns (transitions) for DHAP obtained on exposing cells to 13C6 Glucose for short durations. T1: completely unlabeled form of the molecule (12C), T2: all labeled carbon atoms (13C)

      3. Determination of concentration of each of the transitions: Use the calibration curve to determine the concentration of each of the transition for all the metabolites under study in the test samples. The procedure should be done for all time points post-labeling.
      4. It is important to know the volume of sample prepared (re-suspension volume for the extract from 5 million cells, after drying under N2 gas) and the volume of sample injected into the Mass-spectrometer in order to determine the concentration of the metabolites. The net amount of metabolite in the sample, can be obtained by adding the concentrations of both 13C and 12C transitions
      5. Calculate rate of labeling: For each of the metabolites, determine the rate of label incorporation (or unlabeled pool’s depletion) by calculating the slope of the linear part of the concentration graph , following the below mentioned steps:
      6. By means of Z score by MAD (used for gauging the stability point), determine the stable 13C and 12C concentrations achieved by the isotopic forms after introduction of the label respectively. Formula used for determining the stability point:
        Z-score=(xi - xm)/ MAD; MAD = median{abs(xi -xm)}
        Where xi is the concentration achieved at each time point post labeling by each of the isotopes, and Xm is the median concentration achieved by the metabolite during the labeling time of 60 min.
        This stability point, for 13C isotope is also the saturation concentration achieved by the metabolite.
      7. Once the stability point (stable concentration achieved) is determined, calculate the slope of the curve followed by the metabolite in achieving the stable concentration. This gives the net rate of formation (or degradation).
        The process of rate calculation has been exemplified in the section on representative data.
    2. Determination of percentage of labeled isotopic form for each metabolite
      Calculate the percent label incorporation into each metabolite as follows:
      {(peak area of all isotopic transitions containing 13C labels)/(peak area of all isotopic transitions ie carrying 13C labels+ naturally occurring 13C isotope)}*100

Representative data

The nature of data obtained from kinetic flux profiling experiment, when both, the 13C and 12C isotope, are traced over labeling time, typically looks like the data set in table 1 (plotted in Figure 2).

Figure 2. 13C label incorporation and corresponding reduction in 12C isotope of DHAP upon introduction of labeled media

Table 1. Table representing the values plotted in Figure 1


It can be noted from the data set that in metabolic pathways with high activity, a depletion of the 12C isotope occurs, with a concomitant increase in the 13C isotope quantity. The net amount of the metabolite (a sum of the 13C and 12C concentrations) remains relatively constant over experiments of short durations such as those represented here. This can be used as a thorough check of the data quality since, little or no changes should be expected in total amount of a metabolite under constant conditions during experiments with short labeling times.
Certain metabolic pathways, which are not very active, may not show active depletion of the naturally occurring 12C isotopic pool of their metabolites, in spite of certain amount of 13C labeling. Example1: Calculation of the rate of 13C label incorporation for DHAP (concentration values to be used from Table 1):
Step1: Median (Xm) = 132.28; obtained by calculating the median of 111.44,126.36, 132.28, 136.62, 135.58)
Step 2: Xi-Xm


Table 2. Calculation of Xi-Xm using values from Table 1

Step 3: Abs(Xi-Xm)

Table 3. Calculation of the absolute values of Xi-Xm

Step 4: MAD: Median of Abs(Xi-Xm) = 5.13
Step 5: Z-score

Table 4. Calculation of the Z-score for each concentration value

Notes

  1. During label switch and washes, the media should be constantly maintained at 37 °C. Fluctuations in media temperature cause variations in metabolite profiles.
  2. While seeding cells and labeling, make sure that the incubator surface is uniform and the media is not accumulating in one zone in the culture dish.
  3. During long labeling times (exceeding 1 h), it is advisable to gently swirl the culture dishes every 30 min to aid in uniform distribution of media.
  4. In order to account for cell loss, it is best to seed cells in a parallel plate and perform similar washes as in the lot to be extracted. Instead of adding methanol-water to this set, trypsinize cells and count to get an estimate of the cell number.

Recipes

  1. Cholate buffer
    0.1 M potassium phosphate
    0.05 NaCl
    5 mM cholic acid
    0.1% triton
  2. Quenching mix
    80:20 methanol: water

Acknowledgments

This work was performed as part of the SysTB consortium that is supported by a grant from Department of Biotechnology (DBT)- Government of India. PM is a Senior Research Fellowship recipient from Council of Scientific and Industrial Research-Government of India.

References

  1. Aveldano, M. I. and Horrocks, L. A. (1983). Quantitative release of fatty acids from lipids by a simple hydrolysis procedure. J Lipid Res 24(8): 1101-1105.
  2. Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8): 911-917.
  3. Mehrotra, P., Jamwal, S. V., Saquib, N., Sinha, N., Siddiqui, Z., Manivel, V., Chatterjee, S. and Rao, K. V. (2014). Pathogenicity of Mycobacterium tuberculosis is expressed by regulating metabolic thresholds of the host macrophage. PLoS Pathog 10(7): e1004265.
  4. Sandhoff, R., Brügger, B., Jeckel, D., Lehmann, W. D. and Wieland, F. T. (1999). Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry. J Lipid Res 40(1): 126-132.

简介

哺乳动物细胞中代谢物水平的波动是细胞代谢状态的最直接形式的读出。 当前的协议描述了用于脉冲标记和随后从贴壁哺乳动物细胞分离代谢物的方法。 分离的代谢物可以通过质谱法鉴定和定量,允许估计代谢物从所分析的系统的合成速率和去除速率。

材料和试剂

  1. THP1细胞(来自ATCC,目录号:TIB 202)
  2. 甲醇(LC-MS Ultra CHROMASOLV)(Sigma-Aldrich,目录号:14262)
  3. 水(LC-MS Ultra CHROMASOLV)(Sigma-Aldrich,目录号:14263)
  4. 乙腈(LC-MS Ultra CHROMASOLV)(Sigma-Aldrich,目录号:14261)
  5. 己烷(Sigma-Aldrich,目录号:296090)
  6. HCl(Sigma-Aldrich,目录号:258148)
  7. 台盼蓝(Gibco,目录号:15250-061)
  8. 佛波醇肉豆蔻酸乙酯(PMA)(Sigma-Aldrich,目录号:P8139)
  9. 吡啶(Sigma-Aldrich,目录号:270970)
  10. 三氧化硫吡啶(Sigma-Aldrich,目录号:S7556)
  11. 乙酸钡(Sigma-Aldrich,目录号:243671)
  12. D-葡萄糖( 13 e <99%)(Euriso-Top,目录号:CLM-481)
  13. RPMI培养基1640(Gibco,目录号:31800-014)
  14. 无葡萄糖的RPMI培养基-1640(Sigma-Aldrich,目录号:R1383)
  15. 透析的FBS(Hyclone,目录号:SH30071.03)
  16. 胆固醇缓冲液(见配方)
  17. 淬火混合(参见配方)

设备

  1. 亮线血细胞计数器(Sigma-Aldrich,目录号:Z359629)
  2. 细胞刮刀
  3. 文化菜
    6孔板(Nunc,目录号:140685)
    Flask(T-175,目录号:178983)
  4. 液氮气瓶
  5. 氮气瓶
  6. 水浴
  7. 冰箱(-20,-80,4°C)
  8. 微量离心机(Eppendrof,型号:5418R)
  9. 单螺旋C18塞(GL Sciences,目录号:5010-21700)
  10. HPLC(Agilent,型号:1260 infinity;泵:二元泵VL,型号:G1312C)
  11. HPLC柱
    氨基柱(Polaris 5 NH 2 150 * 2.0mm) C18柱[ZORBAX Eclipse Plus C18(Narrow Bore 2.1 * 150mm5μ)] Cyano柱(Pomenomenax Luna 150 * 2.0mm3μ)
  12. 质谱仪(ABSciex,hybrid 4000 QTrap)
  13. 琥珀色玻璃小瓶(Supelco,目录号:29117)
  14. 玻璃小瓶(Borosil,目录号:9910)
  15. 玻璃牧场移液器
  16. 超声波仪(Branson,型号:1210)

程序

  1. 代谢物标记和提取(Mehrotra等人,2014)
    1. 在所需培养基中培养细胞。对于当前的协议,THP1细胞 在补充有37℃,5%CO 2的10%FCS的RPMI培养基中培养。  计数汇合的细胞(使用台盼蓝)并接种在佛波中 肉豆蔻酸乙酯(PMA)含有用于分化的完全培养基 以80万细胞/培养基完全培养基的密度培养。最后 使用的PMA的浓度为30ng/ml完全培养基和分化  允许48小时。如果希望进行动力学标记,种子 多组细胞,取决于要覆盖的时间点。 例如种子单独的细胞集合以在-0,1,2,5,6,7,8和10天后提取代谢物。 15,30和60分钟的标签介绍。 (PMA分化步骤 是用于THP1细胞。其他贴壁哺乳动物细胞的接种可以是 如常规进行。要接种的细胞数 代谢物提取在质谱仪上获得良好的信号 以针对每种细胞类型进行优化。在我们的情况下获得的信号 来自500万个细胞,接种在6孔板的3个孔中  总共6ml含有完全培养基的PMA。)
      用于标记细胞 与葡萄糖(或任何其它标记物)相比,更换培养基中的培养基 培养皿中补充有10%透析的胎牛血清的新鲜RPMI培养基 血清在引入标记前1小时(重要使用 透析血清作为未透析形式富含小分子 包括葡萄糖)。标记时,从中取出介质 培养皿并用不含葡萄糖的培养基快速洗涤细胞 (整个步骤不应超过30秒 - 使用1-2ml培养基)。这个 步骤是从培养皿中去除未标记的葡萄糖培养基的关键
    2. 加入含有13%葡萄糖的RPMI培养基,补充有10% 透析的胎牛血清加入细胞所需的时间量,  在我们的情况下,从0分钟到60分钟,如所述 更早。对于高达60分钟的孵育,不需要搅拌, 然而对于更长的孵育时间,在断裂中的温和搅拌可以 变得重要。 (葡萄糖浓度-2mg标记的葡萄糖,  ml无葡萄糖的RPM培养基,补充有10%FCS) 介质温度为37°C,因为温度波动可以 诱导代谢轮廓的变化
    3. 完成时 孵育时间,立即取出标记的培养基并加入  淬火混合物冷却(-70℃),甲醇 - 水(80:20) 细胞。对于我们的研究,我们添加了700μl的淬火混合物 足以覆盖接种细胞的表面。没有必要 在加入淬灭混合物之前用无葡萄糖的培养基洗涤细胞 这将仅增加细胞对标记的培养基的暴露时间 (此外,因为我们没有在质谱仪上测量葡萄糖,  剩余的标记携带 13 C葡萄糖的量没有产生任何 差异对我们的观察)。淬火混合物可以掺入  外部标准来衡量提取过程中的任何损失。 (例 的外部标准物是富马酸 - 13 C 14,d 4。其显示峰 从富马酸对应于M + 8的位移。 可以添加标准 到淬火混合物(80:20甲醇:水),然后使用它 代谢猝灭。 标准的浓度需要 优化并且可以在0.1至100ng/ul的淬灭的范围内使用 混合)。
    4. 立即将培养皿置于-75℃下10分钟 以允许完全的代谢猝灭。 接下来是 在冰上孵育10-15分钟以允许冻融细胞
    5. 然后将细胞从干冰上的培养皿上刮下
    6. 涡旋10分钟,30秒的表面活性,然后在冰上孵育1分钟。
    7. 在4℃下以6,000×g离心裂解物5分钟
    8. 收集上清液,加入200μl淬灭混合物到沉淀中 和涡旋硬。 重新旋转管,收集上清液。 重复 步骤。
    9. 将所得的三种上清液汇集(步骤A9-10)
    10. 在N 2气流下干燥上清液。 1,000μl提取物将在约20分钟内干燥
    11. 将干燥的提取物再悬浮于MS级水中(重悬浮 体积取决于细胞数量和MS灵敏度范围。 5 百万THP1细胞,我们使用180μl体积)
    12. 通过样品一次 通过单旋C18柱以除去任何微粒碎片 (这使得LC色谱柱的寿命更长)。
    13. 继续 用于LC-MS分析,样品可以进一步稀释 乙腈或其他有机溶剂,取决于LC色谱柱 要求。 最好分析质量上的分离代谢物   分光仪24小时内隔离。 提取的代谢物应该是 储存于-80℃。 提取物在加工,重悬浮或质量时 分析应保持在4°C。

  2. 脂质(脂肪酸和胆固醇)标记和提取
    1. 脂肪酸和胆固醇标记
      1. 种子和标签单元格 在步骤A14中描述。 对于我们研究中的脂质标记实验, 细胞在携带标记的培养基中温育4小时,不像 代谢物的标记,其与短间隔进行 最大为60分钟。 脂质标记所需的细胞数 实验为3000万,接种在T-175烧瓶中。
      2. 在标签时间结束时,完全从烧瓶中取出标记培养基
      3. 向瓶中加入胆酸盐缓冲液,并在室温下孵育5分钟 温和间歇式攻丝。 对于接种在T-175中的30×10 6个THP1细胞 烧瓶中,使用3ml缓冲液。 要有效收集细胞,请使用a 刮刀。
      4. 收集裂解物并进行硬涡旋(涡流 5分钟,30秒涡旋,然后在冰上孵育30秒) 通过在4℃下3,000×g离心10分钟。
    2. 脂质提取:游离脂肪酸分离
      游离脂肪酸通过遵循Aveldano的方案获得 和Horrocks(1983)略有修改(Aveldano和Horrocks, 1983)。 对于脂质提取和处理,严格的玻璃器皿应该 用过的。 重要的是在这里注意,在脂质提取 并应使用塑料制品
      1. 从中提取脂质   细胞使用Bligh和Dyer方案在Borosil玻璃管(Bligh 和Dyer,1959)。 简而言之,对于在步骤中收集的每1ml裂解物 B1d;加入3.75ml 1:2 CHCl 3:MeOH并充分涡旋一分钟。然后添加 1.25ml CHCl 3并涡旋混合。这之后是最后的添加  的1.25ml水并涡旋。然后将管离心 1,000 rpm,在室温下5分钟,得到两相体系。 小心地提取底层作为含脂区域使用a 玻璃牧场移液管
      2. 在氮气流下干燥提取的脂质,并在-20℃的N 2气体气氛中储存,直到进一步使用。  由于脂质极易发生过氧化反应,因此需要储存干燥的脂质  小瓶顶部充满氮气,在黑暗中。
      3. 将干燥的脂质再悬浮于100μlMS级水中,加入1ml 4:1的乙腈:37%(v/v)盐酸。
      4. 盖上小瓶,并在90°C孵育2小时,以允许酸 水解所有三酰基甘油酯,允许释放脂肪 酸。
      5. 将萃取液冷却至室温,加入1ml己烷,然后涡旋20秒
      6. 将样品在RT下静置5分钟,不受干扰,随后在3,000xg离心5分钟。
      7. 收集上清液,其是具有水解脂质的层。 执行相同的程序将己烷加入到沉淀中并合并   两种上清液
      8. 准确测量收集样品的体积。 将其分成两等份,在氮气流下干燥两组
      9. 干燥后,第一套,加入200μl50:40:5氯仿: 甲醇:水和0.01%氨水,涡旋并直接使用 用于脂质分析 加入20μl的溶液从B3b到含有干燥的小瓶 样本(到在步骤B2j存储的样本)。 样品瓶必须是 。 在水浴中超声处理10秒。
      10. 将样品在室温下放置15分钟。
      11. 孵育完成后,加入2.1μl314 mM乙酸钡溶液。 在水浴中超声处理10秒。
      12. 在RT孵育10分钟,然后在4℃孵育60分钟
      13. 孵育完成后,使小瓶达到室温。 添加120 μl甲醇,并将混合物在13,000×g离心10分钟
      14. 上清液可直接用于胆固醇的质谱测量。

  3. 结果和解释
    随着标记的葡萄糖部分在细胞中代谢,携带标记(由葡萄糖分解代谢形成)的下游代谢物的量增加。 平行地,观察到它们的未标记同位素形式的减少。 该现象可以在质谱仪上捕获。
    基本上有两种数据评估方法:
    1. 绝对代谢物定量和形成/降解速率的测定
      1. 标准曲线:在质量上生成校准曲线 光谱仪,用于待监测的代谢物,通过注射标准   涵盖一系列浓度的解决方案
      2. 测定 代谢物的标记转变:分析质谱数据 从试验样品中获得,仔细识别标记 模式在每个代谢物。例如,当 13 C 6葡萄糖时 喂给THP1细胞,有许多可能的标记模式 (跃迁),可以在质谱仪上获得 二羟基丙酮磷酸盐(DHAP)。然而,如果标记葡萄糖, 通过糖酵解分解代谢,是唯一的主要贡献者 分子,然后在质量上检测到最丰富的跃迁 光谱仪将是完全未标记的形式或一个 携带所有标记的碳

        图1.两个最丰富 在短时间内将细胞暴露于 13 6葡萄糖所获得的DHAP的标记模式(转换)。 T1:完全未标记的 分子( 12 C),T 2:所有标记的碳原子( 13 )

      3. 确定每个转换的浓度:使用 校准曲线来确定每个的浓度 在试验样品中研究的所有代谢物的过渡。 的 应在标记后的所有时间点进行程序
      4. 它是   重要的是要知道样品制备的体积(重悬体积 对于来自500万个细胞的提取物,在N 2气体下干燥后)   注入质谱仪的样品体积 确定代谢物的浓度。 净金额 代谢物,可以通过加入浓度获得 的 13 C和 12 C转换
      5. 计算标签率:For 每种代谢物,确定标记结合的速率(或 未标记的池的消耗)通过计算线性部分的斜率 的浓度图,按照以下步骤:
      6. 通过MAD(用于测量稳定性点)的Z分数, 确定通过同位素实现的稳定的< sup> 13 C和< sup> 12 C浓度  导入标签后的形态。公式用于 确定稳定点:
        Z-score =(xi - xm)/MAD; MAD = median {abs(xi -xm)}
        其中xi是在标记后的每个时间点达到的浓度  ,X m是通过每个同位素实现的中值浓度  代谢物在标记时间为60分钟 对于 13 C同位素,该稳定性点也是代谢物达到的饱和浓度。
      7. 一旦稳定点(实现稳定的浓度) 确定,计算代谢物后面的曲线的斜率 在实现稳定的浓度。这给出净速率 形成(或退化)。
        速率计算的过程已在代表性数据部分中举例说明。
    2. 确定每种代谢物的标记同位素形式的百分比
      计算每种代谢物中标记百分比的百分比如下:
      {(含有 13 C标记的所有同位素跃迁的峰面积)/(峰 面积的所有同位素跃迁,即携带 13 C标记+ 发生的13 C同位素)} * 100

代表数据

当通过标记时间跟踪 13和 12 C同位素时,从动力学通量分析实验获得的数据的性质通常看起来像表中的数据集 1(在图2中绘制) />

13 12 在引入标记培养基后,DHAP的 C同位素

表1.表示图1中绘制的值的表


从数据集可以注意到,在具有高活性的代谢途径中,发生了12 C同位素的耗尽,伴随着13 C同位素量的伴随增加。代谢物的净量(13 C和12 C浓度的和)在短持续时间的实验(例如本文所示的那些)中保持相对恒定。这可以用作对数据质量的彻底检查,因为在短标记时间的实验期间,在恒定条件下代谢物的总量应该很少或没有改变。
尽管有一定量的13 C标记,但某些代谢途径不是非常有活性,它们可能不显示其代谢物的天然存在的12 C同位素库的活性消耗。实施例1:计算DHAP的 13标记结合率(表1中使用的浓度值):
步骤1:中间值(Xm)= 132.28;通过计算111.44,126.36,132.28,136.62,135.58的中值获得)
步骤2:Xi-Xm


表2.使用表1中的值计算Xi-Xm

步骤3:Abs(Xi-Xm)

表3.计算Xi-Xm 的绝对值

步骤4:MAD:Abs(Xi-Xm)的中值= 5.13
第5步:Z分数

表4.计算每个浓度值的Z分数

笔记

  1. 在标签转换和洗涤期间,培养基应该保持在37℃。 介质温度的波动导致代谢物曲线的变化
  2. 在播种细胞和标记时,确保培养箱表面均匀,并且培养基不会在培养皿的一个区域中积累。
  3. 在长标签时间(超过1小时),建议每30分钟轻轻旋转培养皿,以帮助培养基均匀分布。
  4. 为了解决细胞损失,最好将细胞接种在平行板中并进行与待提取的批次相似的洗涤。 而不是添加甲醇 - 水到这组,胰蛋白酶消化细胞和计数得到 估计单元格数量。

食谱

  1. 胆固醇缓冲液
    0.1M磷酸钾
    0.05 NaCl
    5mM胆酸 0.1%triton
  2. 猝灭混合物
    80:20甲醇:水

致谢

这项工作是作为SysTB联盟的一部分,由生物技术部(DBT) - 印度政府的资助。 PM是印度政府科学和工业研究理事会的高级研究员。

参考文献

  1. Aveldano,M.I。和Horrocks,L.A。(1983)。 通过简单的水解程序从脂质定量释放脂肪酸。 Lipid Res 24(8):1101-1105。
  2. Bligh,E.G。和Dyer,W.J。(1959)。 总脂质提取和纯化的快速方法。 Can J Biochem Physiol 37(8):911-917。
  3. Mehrotra,P.,Jamwal,S.V.,Saquib,N.,Sinha,N.,Siddiqui,Z.,Manivel,V.,Chatterjee,S.and Rao,K.V。(2014)。 表达结核分枝杆菌的致病性 通过调节宿主巨噬细胞的代谢阈值。 PLoS Pathog 10(7):e1004265。
  4. Sandhoff,R.,Brügger,B.,Jeckel,D.,Lehmann,W.D.and Wieland,F.T。(1999)。 通过纳米电喷雾离子化串联质谱法测定低皮摩尔水平下的胆固醇。 a> J Lipid Res 40(1):126-132。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mehrotra, P., Saquib, N. and Rao, K. V. (2015). 13C Kinetic Labeling and Extraction of Metabolites from Adherent Mammalian Cells. Bio-protocol 5(8): e1447. DOI: 10.21769/BioProtoc.1447.
  2. Mehrotra, P., Jamwal, S. V., Saquib, N., Sinha, N., Siddiqui, Z., Manivel, V., Chatterjee, S. and Rao, K. V. (2014). Pathogenicity of Mycobacterium tuberculosis is expressed by regulating metabolic thresholds of the host macrophage. PLoS Pathog 10(7): e1004265.
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