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Metabolite and Fatty Acid Analysis of Yeast Cells and Culture Supernatants
酵母细胞和培养上清液中的代谢物和脂肪酸分析   

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Abstract

Metabolite and fatty acid analysis play important roles in evaluating the metabolic state of microorganisms. To examine the growth state and metabolism response of cells to environmental stress or genetic modification, intracellular and extracellular metabolites including fatty acids are usually analyzed to help understand the cellular biochemical changes in microorganisms. In this protocol, gas chromatography-mass spectrometry based analysis was employed to investigate the fatty acids and other metabolites in yeast cells.

Keywords: Metabolite profiling(代谢产物分析), Fatty acid analysis(脂肪酸分析), Saccharomyces cerevisiae(酿酒酵母)

Materials and Reagents

  1. Saccharomyces cerevisiae (S. cerevisiae) cells (EUROSCARF)
  2. Methanol (Sigma-Aldrich, catalog number: 322415 )
  3. Ethanol (Sigma-Aldrich, catalog number: 277649 )
  4. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  5. Acetic acid (Sigma-Aldrich, catalog number: 320099 )
  6. Heptadecanoic acid (Sigma-Aldrich, catalog number: H3500 )
  7. Heptanoic acid (Sigma-Aldrich, catalog number: 75190 )
  8. Ribitol (Sigma-Aldrich, catalog number: A5502 )
  9. Fatty acid methyl esters (FAMEs) mix C8-C24 (Sigma-Aldrich, Supelco, catalog number: 18918 )
  10. 10% BF3-methanol (Fluka, catalog number: 15716 )
  11. Hexane (Sigma-Aldrich, catalog number: 34859 )
  12. SPE column (200 mg/3 ml) (Phenomenex, Strata, model: C18-E )
  13. Adapter (Sigma-Aldrich, Supelco, catalog number: 57020-U )
  14. Disposable syringe (Sigma-Aldrich, catalog number: Z116866 )
  15. Sodium chloride (Sigma-Aldrich, catalog number: 746398 )
  16. Methoxyamine hydrochloride (Sigma-Aldrich, catalog number: 226904 )
  17. N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) (Fluka, catalog number: 69478 )
  18. Agilent Technologies Vial Scr Fix Insert Clr PK100 (Agilent, catalog number: 5188-6591 )
  19. 10 mg/ml heptadecanoic acid (see Recipes)
  20. 10 mg/ml heptanoic acid (see Recipes)
  21. 2 mg/ml ribitol (see Recipes)
  22. Saturated NaCl solution (see Recipes)

Equipment

  1. Glass beads (acid-washed, 425-600 µm) (Sigma-Aldrich, catalog number: G8772 )
  2. Fume hood
  3. SSI 2341-S0S screw cap tubes
  4. 10 ml PP conical bottom tubes
  5. HP-5MS capillary column (30 m x 0.250 mm i.d.; film thickness: 0.25 µm) (Agilent)
  6. Centrifugal Evaporator RC10.09 (Thermo Fisher Scientific, catalog number: 11176780 )
  7. Thermostatic Cabinet Lovibond ET619-4 (Tintometer GmbH, catalog number: ET2428210 )
  8. Vortex mixer (CabNet)
  9. Thermo-shaker
  10. Eppendorf centriguge 5810R (10,000 rpm) (Eppendorf)
  11. Thermo scientific Sorvall Legend Micro 21R centrifuge (12,000 rpm-14,000 rpm) (Thermo Fisher Scientific)
  12. 30 °C Orbital shaker incubator (Yihder Technology, model: LM-570RD )
  13. NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific)
  14. FastPrep® -24 instrument (MP Biomedicals, model: 6004-500 )
  15. Gas chromatography-mass spectrometry (GC-MS) system (Agilent, model: 7890A-5975C ) with an autoinjector G4513A

Software

  1. Agilent MSD Chemstation data analysis software

Procedure

Note: Workflow of metabolite and fatty acid analyses of yeast cells and culture supernatants is shown in Figure 1.

  1. Sample preparation for metabolite and fatty acid analysis
    1. Samples of the yeast culture were collected at different time points and the OD600 was measured using a NanoDrop 2000c spectrophotometer.
      Note: For Saccharomyces cerevisiae cells, OD600 of 1 is roughly equal to 1 x 107 cells/ml. When the OD of cells cultured in medium containing fatty acid is determined, the cells should be washed by 0.9% NaCl solution at least 3 times before OD measurement, since the fatty acid affects the spectral absorbance. If the cells are cultured in YNB or YPD medium, no washing step is needed. Usually, S. cerevisiae cells were cultured at 30 °C.
    2. After centrifugation for 10 min at 10,000 rpm, 4 °C, the supernatant was transferred into a fresh tube by a micropipettor. The cell pellet was resuspended in 0.9% NaCl solution to same OD600.
    3. An equal number of yeast cells (8 x 107) were collected by centrifugation from different yeast cultures and/or time points for metabolite and fatty acid extraction (proceed to step A4). An equal volume of cell culture supernatant (10 ml) was collected for extracellular fatty acid analysis (proceed to step A5).
    4. The cell pellet collected in step A3 was washed twice with cold methanol (< -40 °C) and then collected. The intracellular metabolites and lipids were extracted from control and experimental cells using a chloroform:methanol (2: 1) method adapted from Browse et al. (1986).
      1. The cell pellet was resuspended in 1 ml of 0.9% NaCl solution and then acidified with 200 µl of acetic acid.
      2. As internal standard (IS) to correct for metabolite loss during sample preparation, 10 µl of 10 mg/ml heptadecanoic acid and heptanoic acid dissolved in ethanol and 10 μl of 2 mg/ml ribitol dissolved in water were added to the extraction solvent.
      3. After adding approximately 300 µl of glass beads, the cells were disrupted in a FastPrep®-24 instrument for 30 sec and cooled in ice for 30 sec; this procedure was repeated 4 times.
      4. 3 ml of a chloroform-methanol 2: 1 mixture was added, and the samples were inverted several times, vortexed vigorously, and centrifuged at 10,000 x g for 10 min at 4 °C. The aqueous layer (upper) and cell debris (middle layer) were transferred to a new tube using a micropipetor.
      5. Chloroform layer (lower) was collected. An additional 2 ml of the chloroform was added to the aqueous and cell debris layer to further extract lipids. The generated chloroform layer (lower) was also collected.
      6. The chloroform layer was combined and rotary evaporated to dryness using a Centrifugal Evaporator at 30 °C.
      7. For metabolite profiling, 1 ml of the aqueous phase layer (generated in step A4e) was collected and evaporated at 30 °C overnight to dryness too. The residual aqueous layer and cell debris was discarded.
        Note: Samples need to be completely dry otherwise the remaining water could inhibit the following reaction.
    5. For the fatty acids in the culture medium, the Solid-Phase Extraction (SPE) method (Horak et al., 2009) was adopted.
      1. First, 0.5 ml 1 M HCl, 2 µl 10 mg/ml heptadecanoic acid and 2 µl 10 mg/ml heptanoic acid were added into 10 ml culture supernatant and mixed.
      2. Next, the SPE column was activated with 2.5 ml methanol and washed with 5 ml water. After that, the 10 ml culture supernatant in step A5a was passed through the SPE column.
        Note: Syringe and adapter were used to load liquid onto the SPE column.
      3. The SPE column was washed with 5 ml water and dried at room temperature.
        Note: The SPE column must be dried. Otherwise the reproducibility will be affected.
      4. 1.5 ml chloroform was loaded onto the SPE column for fatty acid elution. The effluent chloroform from the column was collected and also evaporated to dryness. It can be evaporated in a rotary evaporator at 30 °C for several hours or by leaving the tube open in a fume hood overnight.

  2. Fatty acid analysis
    1. Fatty acid derivatization was performed according to previous work (Horak et al., 2009). Fatty acid methyl esters (FAMEs) mix C8-C24 was used as a standard.
      1. The dried lipid residue from step A4f or A5d was redissolved in 500 µl 10% BF3-methanol and incubated in a sealed screw cap tube in a 95 °C heat block for 20 min.
      2. Cool down the tube to near room temperature. 300 µl saturated NaCl in water was added into the reaction, and FAMEs were extracted from the mixture with 300 µl n-hexane. Samples were centrifuged at 14,000 rpm for 1 h at room temperature. The upper hexane layer was next transferred to glass vials for GC-MS analysis.
    2. Chromatography was performed using Agilent Technologies GC-MS system equipped with a HP-5MS capillary column.
      1. Samples of 1 µl were injected into the column by splitless mode using an autoinjector. Helium was used as a carrier gas at 1.1 ml/min. The inlets and MS source temperatures were maintained at 250 and 230 °C, respectively. The oven temperature was maintained at 80 °C for 1 min and ramped to 250 °C at a rate of 7 °C/min, then held at 250 °C for 10 min. Data were acquired in a full scan from 35 to 600 m/z. The representative GC-MS spectra for yeast fatty acid profile is shown in Figure 2.
      2. The detected FAME peaks were integrated after noise reduction and baseline correction. FAME standard curves were made by running different concentrations (0.125-1 mg/ml) of FAMEs mix C8-C24 and IS. The amount of fatty acids were calculated based on extrapolation for the IS and the relative response factors were also calculated. A representative standard curve of C16: 1 m.e. is shown in Figure 3.

  3. Metabolic profiling
    1. The sample for metabolic profiling was derivatized according to previous work (Wang et al., 2010).
      1. The dry residue from step A4g was re-dissolved in 50 µl of 20 mg/ml solution of methoxyamine hydrochloride in pyridine and kept at 37 °C for 60 min for carbonyls protection.
      2. 100 µl of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) was further added to each sample, and silylation was carried out at 70 °C for 30 min.
      3. After incubation, samples were centrifuged at 14,000 rpm for 1 h at room temperature and then transferred to glass vials for GC-MS analysis.
        Note: It would be better that all samples were analyzed within 12 h after they are obtained. The samples can be stored in the sealed vials for a maximum of 2 days at -20 °C. Because the derivative metabolites might be unstable after long term storage, the concentration might decrease.
    2. The same GC-MS system and column (as indicated in step B2) were used.
      1. Samples of 1 µl were injected into the column by splitless mode using an autoinjector. Helium was used as a carrier gas at 1.1 ml/min. The inlets and MS source temperatures were maintained at 250 and 230 °C, respectively. The oven temperature was maintained at 75 °C for 4 min and raised at 4 °C/min to a final temperature of 280 °C and held for 2 min. Mass spectra were recorded from 35 to 600 m/z.
      2. Chromatogram acquisition and mass spectra identification were obtained using the Agilent MSD Chemstation Data Analysis software. Chemical identification of the detected metabolite peaks was performed by searching the NIST08 mass spectral library. The compounds were quantified from the peak area relative to the IS ribitol. No response factors were calculated. The representative GC-MS spectra for yeast metabolic profile are shown in Figure 4.


        Figu
        re 1. Workflow of metabolite and fatty acid analysis of yeast cells and culture supernatants

Representative data



Figur
e 2. The representative GC-MS spectra for a S. cerevisiae fatty acid profile. Fatty acid composition and concentration may differ from those shown here as a result of altered cell culturing conditions and/or alternative genetic backgrounds of the yeast cells employed.


Fi
gure 3. A representative palmitoleic acid methyl ester standard curve. Different concentrations (0.125-0.75 mg/ml) of FAMEs standard were used for GC-MS analysis. To make individual FAME standard curves, the concentration of FAME was determined by mutiplying its percent concentration (as indicated on the cerificate of analysis from the supplier) by the prepared concentrations (0.125-0.75 mg/ml).


Fig
ure 4. A representative GC-MS spectra for L-Alanine derived from total ion chromatograms. Intracellular metabolites were extracted from the S. cerevisiae cells and metabolic profiling was conducted. The metabolite composition and concentration may differ from those shown here as a result of altered cell culturing conditions and/or alternative genetic backgrounds of the yeast cells employed.

Notes

  1. For the metabolite and fatty acid analysis, at least three independent experiments were conducted.
  2. To ensure the reproducibility and comparability, please ensure the detector is well maintained and perform required maintenance for the machine, including cleaning the EI source and replacing the filament as necessary and in accordance with the manufacturers guidelines. For accurate comparisons, it is important that control and experimental cell samples be processed and analyzed in parallel.
  3. Adding or collecting organic solvent was carried out in the fume hood.

Recipes

  1. 10 mg/ml heptadecanoic acid (20 ml)
    Mix 200 mg of heptadecanoic acid with 15 ml ethanol
    Dissolved, add ddH2O to 20 ml
    Stored at 4 °C
  2. 10 mg/ml heptanoic acid
    Mix 217.9 µl heptanoic acid with 15 ml ethanol
    Dissolved, add ddH2O to 20 ml
    Stored at 4 °C
  3. 2 mg/ml ribitol (20 ml)
    Mix 40 mg of ribitol with 15 ml ddH2O
    Dissovled, add ddH2O to 20 ml
    Stored at 4 °C
  4. Saturated NaCl solution
    Mix 35.7 g of NaCl with 80 ml of ddH2O
    Dissolved, add ddH2O to 100 ml
    Stored at room temperature

Acknowledgments

This protocol was adapted from previous work (Chen et al., 2014). This research is supported by a Competitive Research Programme (CRP) grant from the National Research Foundation of Singapore.

References

  1. Browse, J., McCourt, P. J. and Somerville, C. R. (1986). Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152(1): 141-145.
  2. Chen, L., Zhang, J. and Chen, W. N. (2014). Engineering the Saccharomyces cerevisiae β-oxidation pathway to increase medium chain fatty acid production as potential biofuel. PLoS One 9(1): e84853.
  3. Horak, T., Culik, J., Cejka, P., Jurkova, M., Kellner, V., Dvorak, J. and Haskova, D. (2009). Analysis of free fatty acids in beer: comparison of solid-phase extraction, solid-phase microextraction, and stir bar sorptive extraction. J Agric Food Chem 57(23): 11081-11085.
  4. Wang, M., Bai, J., Chen, W. N. and Ching, C. B. (2010). Metabolomic profiling of cellular responses to carvedilol enantiomers in vascular smooth muscle cells. PLoS One 5(11): e15441.

简介

代谢物和脂肪酸分析在评估微生物的代谢状态中起重要作用。 为了检查细胞对环境胁迫或遗传修饰的生长状态和代谢反应,通常分析细胞内和细胞外代谢物(包括脂肪酸)以帮助理解微生物中的细胞生物化学变化。 在本协议中,采用基于气相色谱 - 质谱法的分析来研究酵母细胞中的脂肪酸和其他代谢物。

关键字:代谢产物分析, 脂肪酸分析, 酿酒酵母

材料和试剂

  1. 酿酒酵母 ( 。cerevisiae )细胞(EUROSCARF)
  2. 甲醇(Sigma-Aldrich,目录号:322415)
  3. 乙醇(Sigma-Aldrich,目录号:277649)
  4. 氯仿(Sigma-Aldrich,目录号:C2432)
  5. 乙酸(Sigma-Aldrich,目录号:320099)
  6. 十七烷酸(Sigma-Aldrich,目录号:H3500)
  7. 庚酸(Sigma-Aldrich,目录号:75190)
  8. 核糖醇(Sigma-Aldrich,目录号:A5502)
  9. 脂肪酸甲酯(FAME)混合物C 18 -C 24(Sigma-Aldrich,Supelco,目录号:18918)
  10. 10%BF 3 - 甲醇(Fluka,目录号:15716)
  11. 己烷(Sigma-Aldrich,目录号:34859)
  12. SPE柱(200mg/3ml)(Phenomenex,Strata,型号:C18-E)
  13. 适配器(Sigma-Aldrich,Supelco,目录号:57020-U)
  14. 一次性注射器(Sigma-Aldrich,目录号:Z116866)
  15. 氯化钠(Sigma-Aldrich,目录号:746398)
  16. 甲氧基胺盐酸盐(Sigma-Aldrich,目录号:226904)
  17. 具有1%三甲基氯硅烷(TMCS)(Fluka,目录号:69478)的N-甲基-N-(三甲基甲硅烷基) - 三氟乙酰胺(MSTFA)
  18. Agilent Technologies Vial Scr Fix Insert Clr PK100(安捷伦,目录号:5188-6591)
  19. 10毫克/毫升十七酸(见配方)
  20. 10mg/ml庚酸(参见配方)
  21. 2 mg/ml核糖醇(见配方)
  22. 饱和NaCl溶液(参见配方)

设备

  1. 玻璃珠(酸洗,425-600μm)(Sigma-Aldrich,目录号:G8772)
  2. 通风橱
  3. SSI 2341-S0S螺帽管
  4. 10ml PP锥形底管
  5. HP-5MS毛细管柱(30m×0.250mm i.d .;膜厚度:0.25μm)(Agilent)
  6. 离心蒸发器RC10.09(Thermo Fisher Scientific,目录号:11176780)
  7. 恒温柜Lovibond ET619-4(Tintometer GmbH,目录号:ET2428210)
  8. 涡旋搅拌机(CabNet)
  9. 热振动器
  10. Eppendorf Centriguge 5810R(10,000rpm)(Eppendorf)
  11. Thermo Scientific Sorvall Legend Micro 21R离心机(12,000rpm-14,000rpm)(Thermo Fisher Scientific)
  12. 30℃轨道摇床培养箱(Yihder Technology,型号:LM-570RD)
  13. NanoDrop 2000c分光光度计(Thermo Fisher Scientific)
  14. 涡旋搅拌机(CabNet)
  15. 热振动器
  16. Eppendorf Centriguge 5810R(10,000rpm)(Eppendorf)
  17. Thermo Scientific Sorvall Legend Micro 21R离心机(12,000rpm-14,000rpm)(Thermo Fisher Scientific)
  18. 30℃轨道摇床培养箱(Yihder Technology,型号:LM-570RD)
  19. NanoDrop 2000c分光光度计(Thermo Fisher Scientific)
  20. ... :酵母细胞和培养上清液的代谢物和脂肪酸分析的工作流程如图1所示。

    1. 代谢物和脂肪酸分析的样品制备
      1. 在不同时间点收集酵母培养物的样品,并使用NanoDrop 2000c分光光度计测量OD 600。
        注意:对于Saccharomyc酿酒酵母细胞,OD 600的1大约等于1×10 10 7 > 细胞/ml。当测定在含有脂肪酸的培养基中培养的细胞的OD时,由于脂肪酸影响光谱吸收,因此在OD测量之前应该用0.9%NaCl溶液洗涤细胞至少3次。如果细胞在YNB或YPD培养基中培养,则不需要洗涤步骤。通常,酿酒酵母细胞在30℃培养。
      2. 在10,000rpm,4℃下离心10分钟后,通过微量移液器将上清液转移到新管中。将细胞沉淀重悬于0.9%NaCl溶液中至相同OD 600
      3. 通过离心从不同的酵母培养物和/或代谢物和脂肪酸提取的时间点(进行到步骤A4)收集相等数量的酵母细胞(8×10 7个/孔)。收集等体积的细胞培养物上清液(10ml)用于细胞外脂肪酸分析(进行到步骤A5)
      4. 在步骤A3中收集的细胞沉淀用冷甲醇(<-40℃)洗涤两次,然后收集。使用改编自Browse</em>(1986)的氯仿:甲醇(2:1)方法从对照和实验细胞中提取细胞内代谢物和脂质。
        1. 将细胞沉淀重悬于1ml 0.9%NaCl溶液中,然后用200μl乙酸酸化
        2. 作为样品制备期间校正代谢物损失的内标(IS),将10μl溶于乙醇中的10mg/ml十七烷酸和庚酸和溶于水中的10μl2mg/ml核糖醇加入萃取溶剂中。 br />
        3. 加入约300μl玻璃珠后,将细胞在FastPrep sup-24仪器中破碎30秒,并在冰中冷却30秒;此过程重复4次。
        4. 加入3ml氯仿 - 甲醇2:1混合物,将样品倒置几次,剧烈涡旋,并在4℃下以10,000×g离心10分钟。使用微量移液器将水层(上部)和细胞碎片(中间层)转移到新管中
        5. 收集氯仿层(下层)。 将另外2ml的氯仿加入到水性和细胞碎片层中以进一步提取脂质。 还收集所产生的氯仿层(下层)
        6. 合并氯仿层,用离心蒸发器在30℃下旋转蒸发至干
        7. 对于代谢物分析,收集1ml水相层(在步骤A4e中产生),并在30℃下蒸发过夜至干。 弃去残留的水层和细胞碎片。
          注意:样品需要完全干燥,否则剩余的水会抑制以下反应。
      5. 对于培养基中的脂肪酸,采用固相提取(SPE)方法(Horak等人,2009)。
        1. 首先,将0.5ml 1M HCl,2μl10mg/ml十七烷酸和2μl10mg/ml庚酸加入10ml培养上清液中并混合。
        2. 接下来,用2.5ml甲醇活化SPE柱,并用5ml水洗涤。然后,将步骤A5a中的10ml培养物上清液通过SPE柱 注意:注射器和适配器用于将液体加载到SPE色谱柱上。
        3. 用5ml水洗涤SPE柱,并在室温下干燥。
          注意:SPE柱必须干燥。否则会影响再现性。
        4. 将1.5ml氯仿加载到用于脂肪酸洗脱的SPE柱上。收集来自柱的流出物氯仿,并蒸发至干。它可以在旋转蒸发器中在30℃下蒸发数小时或通过将管在通风橱中开放过夜。

    2. 脂肪酸分析
      1. 根据先前的工作进行脂肪酸衍生化(Horak等人,2009)。使用脂肪酸甲酯(FAME)混合物C 8 -C 24作为标准品。
        1. 将来自步骤A4f或A5d的干燥的脂质残余物再溶解于500μl10%BF 3 - 甲醇中,并在密封的螺旋盖管中在95℃加热块中温育20分钟。
        2. 将管冷却至接近室温。向反应中加入300μl饱和NaCl水溶液,用300μl正己烷从混合物中提取FAME。样品在室温下以14,000rpm离心1小时。接着将上层己烷转移到用于GC-MS分析的玻璃小瓶中
      2. 使用配备有HP-5MS毛细管柱的Agilent Technologies GC-MS系统进行色谱法。
        1. 使用自动注射器通过不分流模式将1μl样品注入柱中。使用氦气作为载气,为1.1ml/min。入口和MS源温度分别保持在250和230℃。烘箱温度在80℃保持1分钟,并以7℃/分钟的速率升温至250℃,然后在250℃保持10分钟。在从35到600m/z的全扫描中获取数据。酵母脂肪酸谱的代表性GC-MS谱如图2所示
        2. 在噪声降低和基线校正后积分检测的FAME峰。通过运行不同浓度(0.125-1mg/ml)的FAME混合物C 18 -C 24和IS制备FAME标准曲线。基于IS的外推计算脂肪酸的量,并计算相对响应因子。 C16的代表性标准曲线:1m.e。如图3所示。

    3. 代谢分析
      1. 根据先前的工作(Wang等人,2010)将代谢分布的样品衍生化。
        1. 将来自步骤A4g的干燥残余物再溶解于50μl20mg/ml甲氧基胺盐酸盐的吡啶溶液中,并在37℃下保持60分钟以保护羰基。
        2. 向每个样品中进一步加入100μl具有1%三甲基氯硅烷(TMCS)的N-甲基-N-(三甲基甲硅烷基) - 三氟乙酰胺(MSTFA),并在70℃下进行甲硅烷基化30分钟。
        3. 孵育后,将样品在室温下以14,000rpm离心1小时,然后转移至玻璃小瓶用于GC-MS分析。
          注意:在获得所有样品后12小时内分析所有样品将是更好的。样品可以在-20℃下在密封的小瓶中储存最多2天。因为衍生代谢物在长期储存后可能不稳定,所以浓度可能会下降。
      2. 使用相同的GC-MS系统和柱(如步骤B2中所示)。
        1. 使用自动注射器通过不分流模式将1μl样品注入柱中。使用氦气作为载气,为1.1ml/min。入口和MS源温度分别保持在250和230℃。烘箱温度在75℃保持4分钟,并以4℃/分钟升至280℃的最终温度并保持2分钟。记录质谱从35至600m/z。
        2. 使用Agilent MSD Chemstation数据分析软件获得色谱图采集和质谱鉴定。通过搜索NIST08质谱库进行检测的代谢物峰的化学鉴定。从相对于IS核糖醇的峰面积量化化合物。没有计算反应因子。酵母代谢图谱的代表性GC-MS谱图如图4所示。


          Figu 1。酵母细胞和培养物上清液的代谢物和脂肪酸分析的工作流程

    代表数据



    Figur e 2。 a的代表性GC-MS光谱。 脂肪酸谱。脂肪酸的组成和浓度可能与这里显示的不同,因为改变的细胞培养条件和/或使用的酵母细胞的替代遗传背景。

    Fi gure 3。代表棕榈油酸甲酯标准曲线。使用不同浓度(0.125-0.75mg/ml)的FAME标准品进行GC-MS分析。为了制备单独的FAME标准曲线,通过将制备的浓度(0.125-0.75mg/ml)乘以其百分比浓度(如来自供应商的分析的证据所示)来确定FAME的浓度。


    图 ure 4。来自总离子色谱图的L-丙氨酸的代表性GC-MS光谱。从S中提取细胞内代谢物。进行了酿酒酵母细胞和代谢谱分析。由于改变的细胞培养条件和/或所用酵母细胞的替代遗传背景,代谢物组成和浓度可能与本文所示不同。

    笔记

    1. 对于代谢物和脂肪酸分析,进行至少三次独立实验
    2. 为确保重现性和可比性,请确保检测器维护良好,并对机器进行必要的维护,包括清洁EI源,必要时更换灯丝以及根据制造商的指南。 为了精确比较,重要的是平行处理和分析对照和实验细胞样品
    3. 在通风橱中进行添加或收集有机溶剂。

    食谱

    1. 10mg/ml十七烷酸(20ml) 将200mg十七烷酸与15ml乙醇混合 溶解后,将ddH 2 O加至20ml ml/h 储存在4°C
    2. 10mg/ml庚酸
      将217.9μl庚酸与15ml乙醇混合 溶解后,将ddH 2 O加至20ml ml/h 储存在4°C
    3. 2mg/ml核糖醇(20ml) 将40mg核糖醇与15ml ddH 2 O混合 Dissovled,将ddH 2 O加到20ml,
      储存在4°C
    4. 饱和NaCl溶液
      将35.7g NaCl与80ml ddH 2 O混合 溶解后,将ddH 2 O加至100毫升
      在室温下贮存

    致谢

    该协议改编自以前的工作(Chen等人,2014)。这项研究由来自新加坡国家研究基金会的竞争研究计划(CRP)资助。

    参考文献

    1. Browse,J.,McCourt,P.J.and Somerville,C.R。(1986)。 在组合消化后确定叶脂质的脂肪酸组成,并从新鲜组织形成脂肪酸甲酯。/a> 152(1):141-145。
    2. Chen,L.,Zhang,J.和Chen,W.N。(2014)。 工程酿造酿酒酵母一个 9(1):e84853。β-氧化途径增加中链脂肪酸生产作为潜在生物燃料。
    3. Horak,T.,Culik,J.,Cejka,P.,Jurkova,M.,Kellner,V.,Dvorak,J.and Haskova,D。(2009)。 啤酒中游离脂肪酸的分析:固相萃取,固相微萃取和 搅拌棒吸着提取。 Agric Food Chem 57(23):11081-11085。
    4. Wang,M.,Bai,J.,Chen,W.N.and Ching,C.B。(2010)。 细胞对血管平滑肌细胞中卡维地洛对映体的细胞反应的代谢组学分析。 PLO 5(11):e15441。
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引用:Chen, L. and Chen, W. N. (2014). Metabolite and Fatty Acid Analysis of Yeast Cells and Culture Supernatants. Bio-protocol 4(17): e1219. DOI: 10.21769/BioProtoc.1219.
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