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Quantification of Bacterial Fatty Acids by Extraction and Methylation
通过提取和甲基化作用量化细菌脂肪酸   

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Abstract

This protocol describes two similar methods for the extraction and methylation of fatty acids from bacterial cultures. The acid derivatization protocol (Lennen et al., 2013; Bligh and Dyer, 1959) results in the extraction and methylation of all fatty acids, both free and bound, from a bacterial culture, while the base derivatization protocol (Lennen and Pfleger, 2013) captures only bound (phospholipid, acyl-thioester) species. After extraction into hexane, the lipids may be analyzed by gas chromatography.

Keywords: Free Fatty Acid(游离脂肪酸), Methylation(甲基化), Fatty Acid Methyl Ester(脂肪酸甲酯)

Materials and Reagents

  1. Cell culture for lipid analysis. When analyzing bacterial cultures expressing a thioesterase, it is advisable to wait until the late stationary phase (8-24 h for E. coli) to perform the extraction to allow sufficient time for product accumulation (Lennen et al., 2013)
  2. Methyl heptadecanoate (should be  ≥ 99% purity) (e.g. Sigma-Aldrich, catalog number: 51633 )
  3. GC sample vials (VWR International, catalog number: 46610-722 ) (ensure you purchase vials compatible with any autosampler you are using)
  4. Compressed nitrogen gas/regulator/tubing
  5. Reverse osmosis water
  6. Methanol
  7. Absolute (100%) ethanol
  8. High resolution gas chromatography grade hexane (99.9% pure mixture of hexane isomers)
  9. Glacial acetic acid (Thermo Fisher Scientific, catalog number: A38-212 )
  10. Deionized water
  11. Anhydrous 1.25 M HCl in methanol (Sigma-Aldrich, catalog number: 17935 )
  12. Sodium bicarbonate
  13. 0.5 M Sodium methoxide in methanol (Sigma-Aldrich, catalog number: 403067 )
  14. Chloroform
  15. Appropriate internal standards (e.g. non-native, odd chain fatty acids). Some possibilities include:
    1. Heptadecanoic acid (C17:0) (99% + pure)
    2. Pentadecanoic acid (C15:0) (99% + pure)
    3. 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine in chloroform (99% + pure) (Avanti Polar Lipids,  catalog number: 850704 )
  16. External standards (the methyl esters of fatty acids you wish to quantify at a known concentration generally these may be obtained from a commercial source). A useful external standard mixture is sold by Supelco through Sigma-Aldrich (catalog number: 18918-1AMP )
  17. Antifoam 204 (Sigma-Aldrich, catalog number: A8311 )

Equipment

  1. 5 ml glass pipettes
  2. 10 ml Glass centrifuge tubes (Thermo Fisher Scientific, KimbleTM, catalog number: 73785-10 ) with fluroropolymer lined caps (Thermo Fisher Scientific, KimbleTM, catalog number: 73802-15415 )
  3. Gloves
  4. Goggles
  5. Appropriate personal protective equipment according to local regulations
  6. Chemical fume hood
  7. Centrifuge
  8. Vortex mixer
  9. Vacuum source/aspiration equipment
  10. Lyophilizer
  11. Water bath
  12. Mass spectrometer or flame ionization detector
  13. Agilent 7890 GC with a 30 m x 0.25 mm HP 5-ms capillary column

Procedure

  1. Collect 2.5 ml of cell culture in a glass centrifuge tube (this volume is sufficient for late exponential/stationary phase cultures of Escherichia coli).
    Note: Most plastic tubes are not compatible with chloroform, which will be added in a subsequent steps.
    Notes:
    1. If you wish to normalize your measured fatty acid concentrations to cell density, record the OD600 at this point.
    2. If your culture is producing free fatty acids, a significant amount of foam may accumulate. It is necessary to collapse the foam, as a significant fraction of the free fatty acids present in the culture may be in the foam. This can be accomplished by the addition of antifoam followed by incubation in an 85 °C water bath for 5-10 min (Lennen et al., 2013). For example, 200 μl of a 1:10 dilution in ethanol of Antifoam 204 was added to collapse the foam of 50 ml shake flask cultures in Lennen et al. 2013.
  2. To the cell culture, add ~50 μg of an appropriate internal standard. Over the course of this procedure, a fraction of this internal standard will be lost. From its known initial concentration and the final amount present, the measured concentration of each fatty acid methyl ester in the final extract can be used to determine the concentration of the corresponding acid in the culture. The choice of internal standard depends on which fatty acids are expected to be abundant in the samples to be analyzed. Often cells do not produce large amounts of odd-chain fatty acids. For example, E. coli grown on glucose does not produce large amounts of odd chain fatty acids, so heptadecanoic acid (C17:0) can be used as an internal standard.
    1. If measuring membrane fatty acids or long chain free fatty acids (C16-C18 species), 5 μl of 10 mg/ml heptadecanoic acid dissolved in ethanol is a useful internal standard.
    2. If the strain of interest is producing large quantities (0.5 g/L or higher) of medium chain free fatty acids (Lennen et al., 2013; Youngquist et al., 2012; Lenen et al., 2010), 50 μl of 10 mg/ml pentadecanoic acid (C15:0) dissolved in ethanol is a useful internal standard.
    3. If only analyzing phospholipid species, a phospholipid internal standard, such as 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine in chloroform, can be used instead of a free fatty acid standard (Lennen and Pfleger, 2013).
    4. Note that all values given for the volume/concentration of the internal standards may need to be adjusted for your particular system, and different standards may need to be used depending on the chain lengths of the fatty acids to be quantified. An internal standard should mimic the compounds to be quantified as closely as possible and should be added to the collected sample of culture in an amount which approximates (same order of magnitude) the concentration of the compounds it will be used to analyze.
    5. 5 μl of 10 mg/ml will correspond to a theoretical final concentration of 50 μg/ml methyl heptadecanoate if two 0.5 ml hexane extractions are performed in step 14. This is around the same order of magnitude as the C14:0, C16:0, and C16:1 fatty acids that are recovered from the membranes of wild-type E. coli.
  3. Add 100 μl of glacial acetic acid to acidify the culture. Carefully vortex the sample to mix. This should be performed under a chemical fume hood.
  4. Add 5 ml of 1:1 mixture by volume of chloroform and methanol with a glass pipette (chloroform will leach a variety of compounds out of a plastic pipette).  Vortex thoroughly.
    Notes:
    1. This mixture may be stored at -80 °C and the extraction (detailed below) finished at a later date if desired.
    2. If samples are frozen, each should be thawed to room temperature before moving on to step 5.
  5. Centrifuge each sample for 10 minutes at 1,000 x g.
  6. Using a vacuum line or aspirator, remove the upper aqueous layer and all cell debris at the interface.
    Notes:
    1. It is acceptable to aspirate a small amount of bottom chloroform layer if internal standards are present.
    2. Prior to aspiration, ensure that proper receptacles are available for the waste generated by this process.
    3. The chloroform layer can be stored at -80 °C at this point if necessary.
    4. This step should be performed under a chemical fume hood!
  7. Evaporate the chloroform extract under a nitrogen stream, leaving a dried residue in the tubes.  
    Notes:
    1. There is usually some residual water that is difficult to remove after this step.
    2. If tubes are being thawed out of the -80 °C freezer, you should re-aspirate any accumulated water droplets before proceeding to the evaporation step.  
    3. This step should be performed under a chemical fume hood!
  8.  Lyophilize the residue (30-60 min is sufficient) to remove any remaining water. All subsequent steps need to be performed with anhydrous samples.

    For Total Fatty Acid Extraction (Free Fatty Acids and Bound Fatty Acid Species by Acid  Catalysis):
  9. To the dried extract, add 0.5 ml of anhydrous 1.25 M HCl in methanol. Cap tightly and heat at 50 °C overnight.
    Notes:
    1. Alternatively, the procedure can be performed at 75-80 °C for 1 h.
    2. The elevated temperature builds up pressure in the sealed vial, so ensure you have tubes capable of withstanding these conditions if you do this (those described in the equipment section are adequate).
    3. This step should be performed under a chemical fume hood!
  10. Cool tubes to room temperature.
  11. Add 0.5 ml of high resolution gas chromatography grade hexane. Take care to work rapidly to recap the tubes as hexane is highly volatile. This step should be performed under a chemical fume hood!
  12. Add 5 ml of a 100 mg/ml NaHCO3 aqueous solution (this concentration is close to the solubility limit–it will dissolve after stirring overnight or warming the solution gently). Addition of bicarbonate quenches the acid-catalyzed reaction. This step should be performed under a chemical fume hood!

    For Bound (Phospholipid) Fatty Acid Extraction by Base Catalysis:
  13. To the dried chloroform extract, add 0.5 ml of 0.5 M sodium methoxide in methanol. This step should be performed under a chemical fume hood!
  14. Incubate the reaction for 10 min at 50 °C, which is sufficient for trans-esterification of common phosphoplipids such as those found in E. coli.  
    Notes:
    1. This reaction can also be used to trans-esterify other bound fatty acids, for example those found in triacylglycerols.
    2. For guidelines on the incubation time for these reactions, which vary from the time required for phospholipids, consult a resource such as Lipid Analysis by William Christie (Christie and Han, 2010). Do not allow the reaction to proceed longer than necessary as too long a reaction time, and the presence of water, can result in esterification of free fatty acids.  
    3. The use of a free fatty acid internal standard as described previously can inform on whether only bound fatty acids are being methylated.  
    4. This step should be performed under a chemical fume hood!
  15. Cool the tubes to room temperature. Quench the reaction by adding 0.1 ml of glacial acetic acid followed by 5 ml of deionized water. This step should be performed under a chemical fume hood!
  16. Add 0.5 ml of high resolution gas chromatography grade hexane. This step should be performed under a chemical fume hood!

    For Both Bound and Total Fatty Acid Extractions:
  17. Vortex tubes thoroughly and centrifuge at room temperature for 10 minutes at 1,000 x g to create a stable interface.  Note that the aqueous phase should be clear before proceeding.
  18. Collect 0.4 ml of the hexane layer from the first extraction in an appropriate gas chromatograph vial. Be sure to cap the vial to prevent sample evaporation. This step should be performed under a chemical fume hood!  
  19. Add 0.5 ml of high resolution gas chromatography grade hexane to the methanol:water extract for a second extraction. Vortex thoroughly to mix and centrifuge at room temperature for 10 minutes at 1,000 x g to create a stable interface. This step should be performed under chemical fume hood!
  20. Collect 0.5 ml of the top hexane layer and add to the hexane collected in step 14. The collected hexane layers are ready for GC analysis.  
    Notes:
    1. Samples may need to be diluted in hexane if fatty acid concentrations are outside the range of standard calibration curves.  
    2. This step should be performed under chemical fume hood!
  21. Analyze by gas chromatography with either a mass spectrometer or flame ionization detector. It is wise to randomize the run order of your samples to prevent the introduction of bias into your analysis. The following is an example of a typical gas chromatography method for bacterial lipids using an Agilent 7890 GC with a 30 m x 0.25 mm HP 5-ms capillary column (Lenen et al., 2010):
    1. Inject 1 μl using a 1:10 split ratio of helium carrier gas (a lower split ratio, e.g. 1:100, can be used for more abundant species).
    2. Oven temperature of 100 °C for 2 minutes.
    3. Oven temperature of 150 °C for 4 minutes.
    4. Ramp to 250 °C at a rate of 4 °C/min.
    If you wish to know more about lipid analysis, an extremely useful reference is the website http://lipidlibrary.aocs.org/index.html. An example with real data is given in step 19.
  22. To determine the concentrations of the fatty acid methyl esters of interest in your chromatography samples, you will need to run samples of these compounds at a variety of known concentrations (your external standards) spanning the full range of concentrations of each compound in the unknown samples. After running these samples, you will have a set of peak areas and corresponding concentrations for each compound. You may then construct a curve of best fit for each compound (including your internal standard), and calculate the concentration of each fatty acid methyl ester and the internal standard in your samples. These concentrations should be multiplied by the ratio of the theoretical to the actual internal standard concentrations to correct for sample lost during the extraction and methylation process. The concentrations in the bacterial culture may then be determined. Keep in mind that the concentration in units of mass for a fatty acid methyl ester and needs to be corrected to report the concentration of the corresponding fatty acid. It is wise to work in molar units during the analysis. As a rule of thumb, you should expect R2 values of about 0.98 to 0.99 or higher from your calibration curves.
  23. Lipids were extracted from a bacterial culture expressing a plant thioesterase specific for 12 carbon (C12) saturated acyl-ACPs, resulting in the overproduction of C12 free fatty acids and, to a lesser extent, C14 free fatty acids. After adding 510 μg of pentadecanoic acid (C15) to 2.5 ml of a late stationary phase culture and performing the total fatty acid extraction protocol by acid catalysis as described above, the resulting FAMEs were analyzed by GC-MS. A total of 1 ml of hexane was used in the extraction. The amount of C12, C14, and C15 FAMEs present in the chromatography sample was determined by constructing calibration curves relating the peak area of each species to its concentration (column 2 of Table 1). If the conversion of C15 acid to its methyl ester and its subsequent extraction were perfect, there would have been 539.5 μg/ml of C15 methyl ester in the chromatography sample (calculated from the listed molecular weights). This fact was used to adjust the concentratiosn of C12 and C14 FAMEs (the measured concentrations were multiplied by the ratio 539.5/484.3). These corrected concentrations were then used to determine the fatty acid titer in the culture by converting to the acid concentration using the molecular weights and then dividing by the volume of the culture sample (2.5 ml).

    Table 1. The titers of C12 and C14 fatty acids in a thioesterase expressing culture were measured by GC-MS using pentadecanoic acid (C15:0) as an internal standard. The concentration refers to the concentration in the sample analyzed by chromatography while titer refers to the concentration in the culture. Molecular weights were obtained from the NIST Chemistry Webbook.


Notes

  1. Chloroform is toxic and should only be used under a fume hood. If chloroform comes into contact with latex or nitrile gloves, remove gloves immediately. Alternatively, use polyvinyl alcohol or SilverShield gloves when working with chloroform.
  2. This procedure uses corrosive (glacial acetic acid and anhydrous hydrochloric acid in methanol), water-sensitive as well as flammable (methanol and hexane) chemicals. Sodium methoxide in methanol is flammable and highly toxic by inhalation, ingestion, and contact with skin. All should be handled according to common chemical safety practices.

Acknowledgments

The protocol described here is based on the work of Bligh and Dyer (1959). The protocol was developed with funding from the US Department of Energy, Great Lakes Bioenergy Research Center.

References

  1. 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.
  2. Christie, W. W. and Han, X. (2010). Lipid analysis: Isolation, separation, identification and lipidomic analysis, Oily Press Bridgewater, UK.
  3. Lennen, R. M. and Pfleger, B. F. (2013). Modulating membrane composition alters free fatty acid tolerance in Escherichia coli. PLoS One 8(1): e54031.    
  4. Lennen, R. M., Politz, M. G., Kruziki, M. A. and Pfleger, B. F. (2013). Identification of transport proteins involved in free fatty acid efflux in Escherichia coli. J Bacteriol 195(1): 135-144.
  5. Lenen, R. M., Braden, D. J., West, R. A., Dumesic, J. A. and Pfleger, B. F. (2010). A process for microbial hydrocarbon synthesis: Overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol Bioeng 106(2): 193-202.
  6. NIST Chemistry WebBook, NIST Standard Reference Database Number 69, Linstrom, P. J. and Mallard, W.G. (eds.) http://webbook.nist.gov/chemistry.
  7. Youngquist, J. T., Lennen, R. M., Ranatunga, D. R., Bothfeld, W. H., Marner, W. D., 2nd and Pfleger, B. F. (2012). Kinetic modeling of free fatty acid production in Escherichia coli based on continuous cultivation of a plasmid free strain. Biotechnol Bioeng 109(6): 1518-1527.

简介

该方案描述了从细菌培养物中提取和甲基化脂肪酸的两种类似方法。 酸衍生化方案(Lennen等人,2013; Bligh和Dyer,1959)导致从细菌培养物中游离和结合的所有脂肪酸的提取和甲基化,而碱衍生化 方案(Lennen和Pfleger,2013)仅捕获结合的(磷脂,酰基 - 硫酯)物质。 在萃取到己烷中之后,可以通过气相色谱分析脂质。

关键字:游离脂肪酸, 甲基化, 脂肪酸甲酯

材料和试剂

  1. 细胞培养用于脂质分析。 当分析表达硫酯酶的细菌培养物时,建议等待直至后期固定期(大肠杆菌的8-24小时)进行提取以允许足够的时间用于产物积累(Lennen et al。,2013)
  2. 十七酸甲酯(纯度> 99%)(例如Sigma-Aldrich,目录号:51633)
  3. GC样品瓶(VWR International,目录号:46610-722)(确保您购买的样品瓶与您使用的任何自动进样器兼容)
  4. 压缩氮气/调节器/管道
  5. 反渗透水
  6. 甲醇
  7. 绝对(100%)乙醇
  8. 高分辨气相色谱级己烷(99.9%纯己烷异构体混合物)
  9. 冰乙酸(Thermo Fisher Scientific,目录号:A38-212)
  10. 去离子水
  11. 无水1.25M HCl的甲醇溶液(Sigma-Aldrich,目录号:17935)
  12. 碳酸氢钠
  13. 0.5M甲醇钠的甲醇溶液(Sigma-Aldrich,目录号:403067)
  14. 氯仿
  15. 适当的内部标准(例如非天然的奇数链脂肪酸)。 一些可能性包括:
    1. 十七酸(C17:0)(99%+纯)
    2. 十五烷酸(C15:0)(99%+纯)
    3. 1,2-二十五酰-sn-甘油-3-磷酸乙醇胺的氯仿溶液(99%+纯)(Avanti Polar Lipids,目录号:850704)
  16. 外部标准(希望在已知浓度下定量的脂肪酸的甲基酯,通常这些可以从商业来源获得)。 有用的外标混合物由Supelco通过Sigma-Aldrich(目录号:18918-1AMP)出售
  17. Antifoam 204(Sigma-Aldrich,目录号:A8311)

设备

  1. 5 ml玻璃移液管
  2. 将具有氟聚合物内衬帽(Thermo Fisher Scientific,Kimble TM ,目录号:73802)的10ml玻璃离心管(Thermo Fisher Scientific,Kimble ,目录号:73785-10) -15415)
  3. 手套
  4. Goggles
  5. 根据当地法规
    处理合适的个人防护装备
  6. 化学通风橱
  7. 离心机
  8. 涡流搅拌器
  9. 真空源/抽吸设备
  10. 冻干机
  11. 水浴
  12. 质谱仪或火焰离子化检测器
  13. Agilent 7890 GC,带有30 m x 0.25 mm HP 5-ms毛细管柱

程序

  1. 在玻璃离心管中收集2.5ml细胞培养物(该体积足以用于大肠杆菌的后期指数/固定相培养物)。
    注意:大多数塑料管与氯仿不兼容,将在后续步骤中添加。
    注意:
    1. 如果您想将测量的脂肪酸浓度标准化为细胞密度,请在此刻记录OD <600> 。
    2. 如果你的文化产生游离脂肪酸,大量的泡沫可能积聚。有必要塌陷泡沫,因为存在于培养物中的游离脂肪酸的显着部分可以在泡沫中。这可以通过加入消泡剂,随后在85℃水浴中孵育5-10分钟来完成(Lennen等,2013)。例如,在Lennen等人中添加200μl的在消泡剂204的乙醇中的1:10稀释液以塌陷50ml摇瓶培养物的泡沫。 2013。
  2. 向细胞培养物中加入〜50μg适当的内标。在该过程的过程中,该内标的一部分将丢失。根据其已知的起始浓度和存在的最终量,最终提取物中每种脂肪酸甲酯的测量浓度可用于测定培养物中相应酸的浓度。内标的选择取决于期望在待分析的样品中富含的脂肪酸。通常细胞不产生大量的奇数链脂肪酸。例如,E。在葡萄糖上生长的大肠杆菌不产生大量的奇数链脂肪酸,因此十七烷酸(C17:0)可以用作内标。
    1. 如果测量膜脂肪酸或长链游离脂肪酸(C 16 16 -C 18物质),5μl溶于乙醇中的10mg/ml十七烷酸是有用的内部标准。
    2. 如果感兴趣的菌株产生大量(0.5g/L或更高)的中链游离脂肪酸(Lennen等人,2013; Youngquist等人, 2012; Lenen等人,2010),将50μl溶解在乙醇中的10mg/ml十五酸(C15:0)用作内标。
    3. 如果仅分析磷脂种类,则可以使用磷脂内标,例如氯仿中的1,2-二十五烷酰基-sn-甘油-3-磷酸乙醇胺代替游离脂肪酸标准(Lennen和Pfleger ,2013)。
    4. 请注意,对于您的特定系统,可能需要调整为内部标准品的体积/浓度给出的所有值,根据要定量的脂肪酸的链长度,可能需要使用不同的标准品。内标应当尽可能接近地模拟待定量的化合物,并且应该以接近(相同的顺序)的量加入收集的培养物样品中 幅度)它将用于分析的化合物的浓度
    5. 如果在步骤14中进行两次0.5ml己烷提取,则5μl10mg/ml将对应于50μg/ml十七烷酸甲酯的理论最终浓度。这与C14:0,C16:0大约相同的数量级,和从野生型E的膜回收的C16:1脂肪酸。大肠杆菌。
  3. 加入100微升冰醋酸酸化培养物。小心涡旋样品混合。这应在化学通风橱下进行
  4. 用玻璃移液管加入5毫升体积比为1:1的氯仿和甲醇混合物(氯仿将从塑料移液管中浸出各种化合物)。彻底涡旋。
    注意:
    1. 该混合物可以储存在-80℃,并且如果需要,提取(下面详述)在稍后的日期完成。
    2. 如果样品已冷冻,则应将每个样品解冻至室温,然后再进行第5步。
  5. 以1,000 x g离心每个样品10分钟。
  6. 使用真空管或吸气器,去除上层水层和界面处的所有细胞碎片 注意:
    1. 如果存在内标物,则可以吸入少量的底部氯仿层。
    2. 在吸入之前,确保为此过程产生的废物提供适当的容器。
    3. 如果需要,氯仿层可以在-80℃下储存。
    4. 此步骤应在化学通风橱下进行!
  7. 在氮气流下蒸发氯仿提取物,在管中留下干燥的残余物。  
    注意:
    1. 通常有一些残留的水,在该步骤后难以除去。
    2. 如果将管从-80°C冰箱中解冻,则在进行蒸发步骤之前,应重新吸入任何积聚的水滴。  
    3. 此步骤应在化学通风橱下进行!
  8.  冻干残余物(30-60分钟就足够了)以除去任何残留的水。 所有后续步骤都需要用无水样品进行
    对于总脂肪酸提取(游离脂肪酸和通过酸催化的结合脂肪酸种类):
  9. 向干燥的提取物中加入0.5ml无水1.25M HCl的甲醇溶液。 盖上盖子,在50℃下加热过夜 注意:
    1. 或者,该程序可以在75-80℃进行1小时。
    2. 升高的温度会在密封的小瓶中产生压力,因此,如果您这样做(设备部分中描述的那些是足够的),请确保您有能够承受这些条件的管。
    3. 此步骤应在化学通风橱下进行!
  10. 将管冷却至室温。
  11. 加入0.5ml高分辨气相色谱级己烷。 由于己烷挥发性高,所以要小心快速地工作。 此步骤应在化学通风橱下进行!
  12. 加入5ml的100mg/ml NaHCO 3水溶液(该浓度接近溶解度极限,在搅拌过夜或温和地温热溶液后,其将溶解)。 加入碳酸氢盐猝灭酸催化的反应。 此步骤应在化学通风橱下进行!

    对于通过碱催化的结合(磷脂)脂肪酸提取:
  13. 向干燥的氯仿提取物中加入0.5ml 0.5M甲醇钠的甲醇溶液。 此步骤应在化学通风橱下进行!
  14. 在50℃下将反应孵育10分钟,这足以用于常见磷脂例如在E中发现的磷脂的酯交换。大肠杆菌。  
    注意:
    1. 该反应也可以用于酯交换其他结合的脂肪酸,例如在三酰基甘油中发现的那些。
    2. 关于这些反应的孵育时间的指导,其不同于磷脂所需的时间,参考例如William Christie的脂质分析(Lipid Analysis)的资源(Christie和Han,2010)。不允许反应进行得比所需更长,因为反应时间太长,并且水的存在可导致游离脂肪酸的酯化。  
    3. 如前所述使用游离脂肪酸内标可以告知是否仅结合的脂肪酸被甲基化。  
    4. 此步骤应在化学通风橱下进行!
  15. 将管冷却至室温。通过加入0.1ml冰醋酸,然后加入5ml去离子水淬灭反应。此步骤应在化学通风橱下进行!
  16. 加入0.5ml高分辨气相色谱级己烷。此步骤应在化学通风橱下进行!

    对于结合和总脂肪酸提取:
  17. 将管彻底涡旋并在室温下以1,000×g离心10分钟以产生稳定的界面。注意,水相在进行之前应该是澄清的。
  18. 在合适的气相色谱小瓶中从第一次萃取中收集0.4ml己烷层。确保盖住样品瓶以防样品蒸发。此步骤应在化学通风橱下进行!  
  19. 向甲醇:水提取物中加入0.5ml高分辨率气相色谱级己烷进行第二次提取。充分涡旋混合并在室温下以1,000×g离心10分钟以产生稳定的界面。这一步应在化学通风橱下进行!
  20. 收集0.5ml的上层己烷层并加入到在步骤14中收集的己烷中。收集的己烷层准备进行GC分析。  
    注意:
    1. 如果脂肪酸浓度超出标准校准曲线的范围,样品可能需要在己烷中稀释。  
    2. 此步骤应在化学通风橱下进行!
  21. 使用质谱仪或火焰离子化检测器通过气相色谱分析。明智的做法是随机排列样品的运行顺序,以防止在分析中引入偏差。以下是使用具有30m×0.25mm HP 5-ms毛细管柱的Agilent 7890 GC(Lenen等人,2010)的用于细菌脂质的典型气相色谱方法的实例:
    1. 使用1:10分流比的氦载气注入1μl(较低的分流比,例如 1:100,可用于更丰富的物种)。
    2. 烘箱温度100℃,2分钟
    3. 烘箱温度150℃,4分钟
    4. 以4℃/min的速率升温至250℃。
    如果您想了解有关脂质分析的更多信息,一个非常有用的参考资料是网站 http://lipidlibrary.aocs。 org/index.html。在第19步中给出了一个有真实数据的例子。
  22. 要确定色谱样品中目标脂肪酸甲酯的浓度,您需要以各种已知浓度(外部标准)运行这些化合物的样品,跨越未知样品中每种化合物的全部浓度范围。运行这些样品后,您将有一组峰面积和每个化合物的相应浓度。然后,您可以构建每个化合物(包括您的内部标准)的最佳拟合曲线,并计算样品中每种脂肪酸甲酯的浓度和内标。这些浓度应乘以理论值与实际内标浓度的比值,以校正萃取和甲基化过程中样品损失。然后可以测定细菌培养物中的浓度。请记住,脂肪酸甲酯的质量单位的浓度,需要校正以报告相应脂肪酸的浓度。在分析期间以摩尔单位工作是明智的。作为一个经验法则,你应该期望 R 2 值约为0.98至0.99或更高
  23. 从表达对12个碳(C12)饱和的酰基-ACP具有特异性的植物硫酯酶的细菌培养物中提取脂质,导致C12亚游离脂肪酸和在较小程度上过量产生C14游离脂肪酸。在如上所述通过酸催化将510μg十五烷酸(C15)加入2.5ml后固定相培养物中并进行总脂肪酸提取方案后,通过GC-MS分析所得的FAME。在萃取中使用总共1ml的己烷。通过构建将每种物质的峰面积与其浓度(表1的第2列)相关的校准曲线,确定色谱样品中存在的C12,C14和C15 FAME的量。如果C15酸向其甲酯的转化及其随后的提取是完美的,则在色谱样品中将存在539.5μg/ml的C15甲酯(从列出的分子量计算)。该事实用于调节C12和C14FAME的浓度(测量的浓度乘以比率539.5/484.3)。然后使用这些校正的浓度通过使用分子量转换为酸浓度,然后除以培养物样品的体积(2.5ml)来确定培养物中的脂肪酸滴度。

    表1.使用十五烷酸(C15:0)作为内标通过GC-MS测量表达硫酯酶的培养物中的C12和C14脂肪酸的滴度。通过色谱法分析样品,而滴度指培养物中的浓度。分子量从NIST化学书籍获得。


笔记

  1. 氯仿有毒,只能在通风橱下使用。 如果氯仿与胶乳或丁腈手套接触,请立即脱下手套。 或者,在使用氯仿时,请使用聚乙烯醇或SilverShield手套。
  2. 此程序使用腐蚀性(冰醋酸和甲醇中的无水盐酸),水敏感性以及易燃(甲醇和己烷)化学品。 甲醇钠在甲醇中是易燃的,通过吸入,摄入和与皮肤接触具有高毒性。 所有这些都应根据通用的化学品安全规范进行处理。

致谢

这里描述的协议是基于Bligh和Dyer(1959)的工作。 该协议是由美国能源部,大湖生物能源研究中心资助开发的。

参考文献

  1. 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
  2. Christie,W. W.和Han,X。(2010)。脂质分析:分离,分离,鉴定和脂质组学分析,Oily Press Bridgewater,UK
  3. Lennen,R. M.和Pfleger,B. F.(2013)。 调节膜组成改变大肠杆菌中的游离脂肪酸耐受性。 a> PLoS One 8(1):e54031。    
  4. Lennen,R.M.,Politz,M.G.,Kruziki,M.A.and Pfleger,B.F。(2013)。 在大肠杆菌中鉴定与游离脂肪酸流出有关的转运蛋白。 J Bacteriol 195(1):135-144。
  5. Lenen,R.M.,Braden,D.J.,West,R.A.,Dumesic,J.A。和Pfleger,B.F。(2010)。 微生物碳氢化合物合成的过程:大肠杆菌中的脂肪酸过量生产 和催化转化为烷烃。 Biotechnol Bioeng 106(2):193-202
  6. NIST化学WebBook,NIST标准参考数据库编号69,Linstrom,PJ和Mallard,WG(eds。) http://webbook.nist.gov/chemistry
  7. Youngquist,J.T.,Lennen,R.M.,Ranatunga,D.R.,Bothfeld,W.H.,Marner,W.D.,2nd和Pfleger,B.F。(2012)。 基于连续培养的大肠杆菌中的游离脂肪酸生产的动力学模型 的质粒自由菌株。 Biotechnol Bioeng 109(6):1518-1527。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Politz, M., Lennen, R. and Pfleger, B. (2013). Quantification of Bacterial Fatty Acids by Extraction and Methylation. Bio-protocol 3(21): e950. DOI: 10.21769/BioProtoc.950.
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kamran Jawed
ICGEB
If I want to extract FAME from small volume of cell culture (100-500 ul), how can I modify this protocol.
4/11/2015 5:02:25 AM Reply
Bio-protocol Editorial Team
bio-protocol.org

Authors indicated that they did not try to use that small volume of cell culture for this protocol, thus they did not have good answer to your question.
Please share your experience with other readers via our "Reproducibility Feedback" if you successfully modify this protocol for that smaller vol. of cell culture in the future.

Good luck,

Bio-protocol team

5/20/2015 2:06:38 PM