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Determining Efficiency and Selectivity of Lipid Extraction by Perturbing Agents from Model Membranes
采用源自模型膜的扰动剂测定脂质提取的效率和选择性   

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Abstract

Several membrane-perturbing agents extract lipids from membranes and, in some cases, this lipid efflux is lipid specific. In order to gain a better description of this phenomenon and to detail the intermolecular interactions that are involved, a method has been developed to characterize the extent and the specificity of membrane-lipid extraction by perturbing agents. A perturbing agent is incubated with model membranes existing as multilamellar vesicles (MLVs) and subsequently, the remaining MLVs and the small lipid/perturbing agent complexes resulting from the extraction are isolated and analysed to assess the extent and the specificity of the lipid extraction.

Background

Several membrane-perturbing agents extract lipids from membranes; these include proteins, peptides, and detergents. Several of these lipid extractions are fundamental processes in biology. In some cases, the process is lethal; this is the case for some antibacterials that extract lipids from bacterial membranes leading to the death of the cells (Bechinger, 2014; Schaefer et al., 2014). In other systems, this process is vital. For example, ApoA1 is a protein that binds to cells and extracts phospholipids and cholesterol to form nascent high density lipoproteins (HDL), a process critical for the reverse cholesterol transport and therefore playing a pivotal role in the control of atherosclerosis (Strömstedt et al., 2010). It has been shown that some of these lipid extractions are lipid specific; in other words, the lipid composition of the extracted fraction is different than that of the original membranes. It is expected that such specificity will be reported more often considering the recent progress of lipidomics. In general, the lipid specificity of the induced lipid efflux is poorly characterized despite the pivotal role it plays in biological processes. We have recently shown that selective lipid efflux depends on specific interactions with membranes (Therrien et al., 2013; 2016). In order to gain a better description of this emerging phenomenon and to detail the intermolecular interactions that are involved, a method has been developed to characterize the extent and the specificity of membrane-lipid extraction by perturbing agents using model membranes. A perturbing agent is incubated with model membranes existing as multilamellar vesicles (MLVs). The classes of perturbing agents include proteins (e.g., binder-of-sperm proteins BSPA1, ApoA1), peptides (e.g., melittin, antibacterial peptides), and detergents (e.g., Triton X-100). The use of model membranes allows a complete control of the lipid composition, modulating in a precise manner molecular features such as the charge and the H-bond capacity, and defining how these factors contribute to the lipid specificity of the extraction. Subsequently, the remaining MLVs, which may include some perturbing agent, are separated by centrifugation from the small lipid/perturbing agent complexes resulting from the lipid extraction. The lipid compositions of the pellet (representative of intact membranes) and of the supernatant (representative of the lipid efflux) are analysed in order to assess the extent and the specificity of the lipid extraction.

Materials and Reagents

  1. Progene® 1.5-ml microcentrifuge tubes
  2. V-Vial screw-thread sample vials, 5 ml, with PTFE-lined caps
  3. Pyrex Brand 9800 test tubes, 20 ml
  4. Syringe
  5. Glass test tube
  6. Marbles
  7. YMC diol column
  8. Lipids (high purity) (Avanti Polar Lipids) (ex. 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine [POPC]; 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine [POPE])
  9. Membrane perturbing agent of interest
  10. Benzene (ACS grade)
  11. Methanol (HPLC grade)
  12. Milli-Q water
  13. 3-[N-morpholino]propanesulfonic acid (MOPS) (> 95%)
  14. Sodium chloride (NaCl, high-purity grade)
  15. Ethylenediamine tetraacetic acid (EDTA) (99.4-100.6%)
  16. Potassium phosphate, monobasic (KH2PO4) (dried at 105 °C under vacuum for at least 4 h prior to its use to ensure it is dry)
  17. Concentrated sulphuric acid (H2SO4) (ACS reagent, 95.0-98.0%)
  18. Peroxide (H2O2) (30%) (ACS reagent)
  19. Sodium metabisulfite (Na2S2O5) (ACS reagent, >97%)
  20. Ammonium molybdate tetrahydrate [(NH4)6Mo7O24·4H2O] (ACS reagent, 81.0-83.0%)
  21. Ascorbic acid (99%)
  22. Acetonitrile (HPLC grade)
  23. Ammonium acetate (CH3CO2NH4) (≥ 98%)
  24. Nitrogen

Equipment

  1. Microcentrifuge (Eppendorf, model: 5417 R ) equipped with a rotor (Eppendorf, model: f45-30-11 )
  2. UV-Vis spectrometer (Agilent Technologies, model: Cary 6000i )
  3. LC/MS system (Agilent Technologies, model: 1100 series system ) with a 1100 MSD mass spectrometer (Agilent Technologies, model: 1100 MSD )
  4. Water bath

Procedure

Typically, a series of experiments is planned for a given perturbing agent and model membranes, at different ratios of lipid to perturbing agent. Samples are carried out in triplicates. Stock solutions are prepared to perform one set of measurements. For a single measurement, between 300 and 800 nmoles of phospholipids are needed; the lipid amount is kept constant in all samples.

  1. Model membrane preparation
    1. To form model membranes from a binary mixture of lipids, individual lipids are first dissolved in a benzene/methanol mixture. Typically an individual lipid stock solution is prepared by weighting exactly ~25 mg of lipid and by adding the appropriate volume of benzene/methanol (90/10, v/v) solution to obtain 5 mg of lipid/ml. For more polar species, such as phosphatidylserine, and cholesterol sulphate, the proportion of methanol can be increased to 85/15 (v/v). The lipid organic solutions are prepared in V-Vial screw-thread sample vials, 5 ml, in order to limit solvent evaporation.
    2. Aliquots with appropriated volumes of lipid solutions are mixed to obtain the desired molar ratio. The absolute amount of lipid depends on the number of planned samples.
    3. The lipid solution is then frozen in liquid nitrogen and lyophilized for at least 16 h to make sure that all the organic solvent is removed.
    4. The lipid powder is hydrated in a MOPS buffer (50 mM) containing 100 mM NaCl and 100 µM EDTA, and adjusted to pH 7.4. Typically the appropriate buffer volume is added to provide a MLV stock suspension of 5 mM.
    5. The samples are submitted to 3 freeze-and-thaw cycles (from liquid nitrogen temperature to a temperature about 10 °C above the temperature of the gel-to-fluid phase transition). The sample is vortexed when the sample is at high temperature to ensure the proper hydration of the lipids.
    Note: Other buffers could be used but they must be phosphate free in order to assay phospholipids.
    Example: model membrane preparation
    Membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-phosphoethanolamine (POPE) with a POPC/POPE molar ratio of 60/40; sufficient lipid amount for 10 samples.
    Molecular weight (POPC) = 760.07 g/mol; Molecular weight (POPE) = 718.01 g/mol
    Organic solution: 5 mg of phospholipid/ml.
    730 µl of POPC organic solution (3.65 mg; 4.80 µmol) were mixed with 460 µl of POPE organic solution (2.30 mg; 3.20 µmol).
    After lyophilization, the resulting lipid powder (5.95 mg; 8.00 µmol) was hydrated with 1.6 ml of buffer to provide a MLV stock suspension of 5 mM.

  2. Lipid extraction
    The lipid extraction is determined for a given membrane composition, a given temperature, and a given concentration of perturbing agent. All the samples, including the controls (i.e., without the perturbing agent), have the same final volume, and final lipid concentration.
    1. Each sample is a combination of an aliquot of MLV suspension, an aliquot of perturbing agent solution, and an aliquot of buffer. The final concentration of lipid is set between 1.5 and 4 mM, and the concentration of perturbing agent depends on the desired lipid/perturbation agent molar ratio. The total volume should be between 250 and 1,500 µl. The appropriate volume of MLV stock suspension is pipetted in a 1.5 ml centrifuge tube. The aliquot of perturbing agent solution, prepared in the same buffer as the lipid suspension, is added to obtain the desired lipid/perturbing agent ratio. An aliquot of buffer is added to ensure that all samples have the same volume and the same lipid concentration (Table1).
      Table 1 shows an example of the composition of a sample series to determine the efficiency and selectivity of lipid extraction induced by a detergent. In this case, the concentration of the lipid stock solution is 40 mM while the concentration of the detergent stock solution is 10 mM.

      Table 1. Example of sample series: Lipid extraction by a detergent


    2. The suspensions are then vortexed and incubated for at least 30 min at a given temperature, using a water bath.
    3. After the incubation, the samples are centrifuged for 5 min at 20,800 x g and 1 °C. The extracted lipids are defined as those existing in small perturbing agent/lipid assemblies that stay in the supernatant while the remaining MLVs (possibly with bound perturbing agent) pellet. Centrifugation of control samples (without perturbing agent) typically show that more than 95% of lipids are found in the pellets.
      Note: Another temperature can be selected for the centrifugation; the control samples (without perturbing agent) provide a quantitative description of the efficiency of the separation of the extracted lipids from the remaining MLVs by centrifugation.
    4. The supernatants are isolated using a syringe.
    5. The pellets are resuspended in 500 µl of MOPS buffer for their analysis.

  3. Lipid analysis
    Two parallel analyses are carried out to determine the extent of lipid extraction. First, the phospholipid contents in the supernatants, and in the pellets are determined by a Bartlett phosphorus assay (Bartlett, 1958). Second, an LC-MS analysis is carried out for the supernatants and the pellets.

  4. Bartlett phosphorus assay
    1. An aliquot of the lipid sample containing between 8 and 70 nmol in phospholipid is transferred into a glass test tube. Typically, 20 µl of the supernatant and 20 µl of resuspended pellet are used.
    2. Standard samples containing between 0 (blank), and 3.5 mM phosphate are prepared in glass test tubes, from a KH2PO4 stock solution (3.5 mM in Milli-Q water; 47.63 mg in 100 ml). Typically, samples with 3.5, 2.0, 1.5, 1.0, 0.4 mM KH2PO4 are prepared.
    3. A 20-µl aliquot of each standard sample is transferred into a glass test tube.
    4. To each tube, 120 µl of concentrated sulphuric acid is added. The samples are then vortexed.
    5. To each tube, 20 µl of H2O2 (30%) is added. The samples are then vortexed.
    6. The tubes are heated all together to 200 °C for 10 min. During the heating, marbles are placed on top of the tubes to prevent potential evaporation.
    7. The samples are put on the bench and left to cool to room temperature.
    8. To each tube, 1,340 µl of Milli-Q water is added. The samples are then vortexed.
    9. To each tube, 40 µl of Na2S2O5 solution (526 mM in Milli-Q water; 100 mg in 1 ml) is added. The samples are then vortexed.
    10. The tubes are heated all together to ~90 °C for exactly 5 min.
    11. The samples are put on the bench and left to cool to room temperature.
    12. To each tube, 400 µl of (NH4)6Mo7O24·4H2O solution (16.2 mM in Milli-Q water; 200 mg in 10 ml) is added. The samples are then vortexed.
    13. To each tube, 40 µl of ascorbic acid (568 mM in Milli-Q water; 100 mg in 1 ml – the solution is photosensitive and must be kept in the dark prior its use) are added. The samples are then vortexed.
    14. The tubes are heated all together to ~90 °C for exactly 10 min. A blue color should appear.
    15. The samples are put on the bench and left to cool to room temperature.
    16. The absorbance at 820 nm is read within 1 h and is used for the phospholipid determination (Figure 1).


      Figure 1. Typical calibration curve for the Bartlett assay

  5. LC-MS analysis
    The lipid composition of the supernatants and of the pellets are determined by LC-MS analysis. Typically 32-µl aliquots of each sample are injected and triplicates are carried out for each condition. Samples are eluted on a YMC diol column (4.6 x 150 mm, 5 µm particle size) (Agilent Technologies), maintained at 50 °C. Elution of the phospholipids is achieved in 7 min, using acetonitrile/aqueous ammonium acetate solution (100 mM) (85/15, v/v) at 0.6 ml/min. The ESI source is used in the positive ionization mode. Nitrogen is used as drying gas at 250 °C and 12 L/min. Nebulizing gas is also nitrogen, held at 241 kPa.
    1. A 2-µl aliquot of the supernatant and of the resuspended pellet is diluted in 1 ml of an H2O/MeOH (1/9, v/v) solution.
    2. A 2-µl aliquot is injected for the LC/MS analysis.

Data analysis

  1. The Bartlett assay
    The fraction of extracted phospholipids, f, is provided by:

  2. The LC/MS analysis
    The extent of extraction for each lipid species is determined using:
    Extraction% = As/(As + Ap) x 100%
    Where,
    As and Ap are the lipid peak area from the supernatant and the pellet analysis, respectively. The lipid specificity of the extraction is obtained by comparing Extraction% calculated for each lipid. In the absence of specificity, Extraction% should be the same for the different lipids. A higher Extraction% factor for one lipid relative to the other defines its specific extraction.

    Example: An example of results showing some lipid selectivity for a detergent-induced extraction of phosphatidylcholine (PC)/phosphatidylethanolamine (PE) membranes is presented in Table 2.

    Table 2. Example of sample series: Selectivity of lipid extraction by a detergent. The lipid concentration was constant (1.5 mM) and the detergent concentration is varied.

Notes

  1. For the Bartlett assay, the reproducibility for independent triplicates was typically 8% and better than 10%.
  2. For the LC/MS analysis, the reproducibility between the 3 injections of a sample is typically better than 1%, and it is better than 5% for independent triplicates.
  3. For detergent concentration between 0.75 and 4 mM, the extraction is PC specific as more PC are extracted relative to PE. When the MLVs are intact or completely disrupted, lipid extraction specificity cannot be expressed.
  4. The agreement of the total phospholipid extraction between the LC/MS and the Bartlett assay reflects the accuracy of the determination.

Acknowledgments

This work was supported by the Natural Sciences and Engineering Research Council of Canada, and by the Fonds de recherche du Québec-Nature et technologies through its Strategic Cluster program. The protocol is adapted from previous work (Therrien et al., 2013; 2016).

References

  1. Bartlett, G. R. (1958). Phosphorous assay in column chromatography. J Biol Chem 234: 466-468.
  2. Bechinger, B. (2014). The SMART model: Soft membranes adapt and respond, also transiently, in the presence of antimicrobial peptides. J Peptide Sci 21: 346-355.
  3. Schaefer, E. J., Anthanont, P. and Asztalos, B. F. (2014). High-density lipoprotein metabolism, composition, function, and deficiency. Curr Opin Lipidol 25:194-199.
  4. Strömstedt, A. A., Ringstad, L., Schmidtchen, A. and Malmsten, M. (2010). Interaction between amphiphilic peptides and phospholipid membranes. Curr Opin Colloids Interface Sci 15:467-478.
  5. Therrien, A., Fournier, A. and Lafleur, M. (2016). Role of the cationic C-terminal segment of melittin on membrane fragmentation. J Phys Chem B 120(17): 3993-4002.
  6. Therrien, A., Manjunath, P. and Lafleur, M. (2013). Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1. Biochim Biophys Acta 1828(2): 543-551.

简介

几种膜干扰剂从膜提取脂质,并且在一些情况下,该脂质外排是脂质特异性的。为了更好地描述这种现象并详细描述所涉及的分子间相互作用,已经开发了一种方法来表征通过扰动剂提取膜 - 脂质的程度和特异性。将扰动剂与作为多层囊泡(MLV)存在的模型膜一起孵育,随后分离并分析从提取得到的剩余MLV和小脂质/扰动剂复合物,以评估脂质提取的程度和特异性。 br />
[背景] 几种膜干扰剂从膜中提取脂质;这些包括蛋白质,肽和洗涤剂。这些脂质提取中的几种是生物学中的基本过程。在某些情况下,该过程是致命的;这是从细菌膜提取脂质导致细胞死亡的一些抗菌剂的情况(Bechinger,2014; Schaefer等人,2014)。在其他系统中,这个过程是至关重要的。例如,ApoA1是结合细胞并提取磷脂和胆固醇以形成新生高密度脂蛋白(HDL)的蛋白质,这是反向胆固醇转运的关键,因此在控制动脉粥样硬化中起关键作用(Strömstedt et al 。,2010)。已经表明,这些脂质提取物中的一些是脂质特异性的;换句话说,提取级分的脂质组成不同于原始膜的脂质组成。预期这种特异性将更经常地考虑脂质组学的最近进展而报告。一般来说,这些诱导的脂质流出的脂质特异性很差地表征,尽管它在生物过程中起关键作用。我们最近已经显示选择性脂质流出取决于与膜的特异性相互作用(Therrien等人,2013; 2016)。为了更好地描述这种出现的现象并详细描述所涉及的分子间相互作用,已经开发了一种方法来表征通过使用模型膜的扰动剂提取膜 - 脂质的程度和特异性。扰动剂与作为多层囊泡(MLV)存在的模型膜孵育。扰动剂的类别包括蛋白质(例如,精子蛋白BSPA1,ApoA1的结合物),肽(例如,蜂毒肽,抗菌肽)和洗涤剂,例如。,Triton X-100)。模型膜的使用允许完全控制脂质组成,以精确的方式调节分子特征例如电荷和H键能力,并且限定这些因子如何有助于提取的脂质特异性。随后,可以包括一些扰动剂的剩余MLV通过离心从由脂质提取产生的小脂质/扰动剂复合物中分离。分析沉淀(代表完整膜)和上清液(代表脂质流出)的脂质组成,以评估脂质提取的程度和特异性。

材料和试剂

  1. Progene ® 1.5 ml微量离心管
  2. V-Vial螺纹样品瓶,5 ml,带PTFE内衬盖
  3. Pyrex Brand 9800试管,20ml
  4. 注射器
  5. 玻璃试管
  6. 大理石
  7. YMC二醇柱
  8. 脂质(高纯度)(Avanti Polar Lipids)(例如1-棕榈酰-2-油酰-sn-甘油基 - 磷酸胆碱[POPC]; 1-棕榈酰-2-油酰-sn-甘油基 - 磷酸乙醇胺[POPE] >
  9. 感兴趣的膜扰动剂
  10. 苯(ACS级)
  11. 甲醇(HPLC级)
  12. Milli-Q水
  13. 3- [N-吗啉代]丙磺酸(MOPS)(> 95%)
  14. 氯化钠(NaCl,高纯度级)
  15. 乙二胺四乙酸(EDTA)(99.4-100.6%)
  16. 磷酸二氢钾(KH 2 PO 4)(在使用前在105℃真空干燥至少4小时以确保其干燥)
  17. 浓硫酸(H 2 SO 4)(ACS试剂,95.0-98.0%)
  18. 过氧化物(H 2 O 2)(30%)(ACS试剂)
  19. 偏亚硫酸氢钠(Na 2 SS 2 O 5)(ACS试剂,> 97%)
  20. 钼酸铵四水合物[(NH 4)6 Mo 6 Mo 7 SO 4·4H 2·sub > O](ACS试剂,81.0-83.0%)
  21. 抗坏血酸(99%)
  22. 乙腈(HPLC级)
  23. 乙酸铵(CH 3 CO 2 NH 4)(≥98%)
  24. 氮气

设备

  1. 装备有转子(Eppendorf,型号:f45-30-11)的微量离心机(Eppendorf,型号:5417R)
  2. UV-Vis光谱仪(Agilent Technologies,型号:Cary 6000i)
  3. 使用1100MSD质谱仪(Agilent Technologies,型号:1100MSD)的LC/MS系统(Agilent Technologies,型号:1100系列系统)
  4. 水浴

程序

通常,对于给定的扰动剂和模型膜,在脂质与扰动剂的不同比例下计划一系列实验。样品一式三份进行。制备储备溶液以进行一组测量。对于单次测量,需要300至800nmol的磷脂;脂质量在所有样品中保持恒定。

  1. 模型膜制备
    1. 为了由脂质的二元混合物形成模型膜,首先将单个脂质溶解在苯/甲醇混合物中。通常,通过精确称重〜25mg脂质并通过加入适当体积的苯/甲醇(90/10,v/v)溶液以获得5mg脂质/ml来制备单个脂质储备溶液。对于更极性的物质,例如磷脂酰丝氨酸和胆固醇硫酸盐,甲醇的比例可以增加到85/15(v/v)。在V-Vial螺纹样品瓶(5ml)中制备脂质有机溶液,以限制溶剂蒸发。
    2. 将具有适当体积的脂质溶液的等分试样混合以获得所需的摩尔比。脂质的绝对量取决于计划样品的数量。
    3. 然后将脂质溶液在液氮中冷冻并冻干至少16小时,以确保除去所有有机溶剂。
    4. 脂质粉末在含有100mM NaCl和100μMEDTA的MOPS缓冲液(50mM)中水合,并调节至pH 7.4。通常加入适当的缓冲液体积以提供5mM的MLV原液悬浮液
    5. 将样品进行3个冷冻 - 解冻循环(从液氮温度至高于凝胶 - 流体相转变温度约10℃的温度)。当样品处于高温时涡旋样品以确保脂质的适当水合。
    注意:可以使用其他缓冲液,但为了测定磷脂,它们必须不含磷酸盐。
    示例:模型膜制备
    由具有POPC/POPE摩尔比为60/40的1-棕榈酰-2-油酰-sn-甘油磷酸胆碱(POPC)和1-棕榈酰-2-油酰-sn-甘油基 - 磷酸乙醇胺(POPE)制成的膜;足够的脂质量为10个样品。
    分子量(POPC)= 760.07g/mol;分子量(POPE)= 718.01g/mol
    有机溶液:5mg磷脂/ml。
    将730μlPOPC有机溶液(3.65mg;4.80μmol)与460μlPOPE有机溶液(2.30mg;3.20μmol)混合。
    冻干后,所得脂质粉末(5.95mg;8.00μmol)用1.6ml缓冲液水合,得到5mM的MLV储备液。

  2. 脂质提取
    对于给定的膜组合物,给定的温度和给定浓度的扰动剂,确定脂质提取。所有样品,包括对照(即,没有扰动剂),具有相同的最终体积和最终脂质浓度。
    1. 每个样品是MLV悬浮液的等分试样,扰动剂溶液的等分试样和缓冲液的等分试样的组合。脂质的最终浓度设定在1.5和4mM之间,并且扰动剂的浓度取决于所需的脂质/扰动剂摩尔比。总体积应在250和1500μl之间。将合适体积的MLV储备悬浮液移液到1.5ml离心管中。加入在与脂质悬浮液相同的缓冲液中制备的扰动剂溶液的等分试样,以获得所需的脂质/扰动剂比例。加入一份缓冲液以确保所有样品具有相同的体积和相同的脂质浓度(表1)。
      表1显示了用于确定洗涤剂诱导的脂质提取的效率和选择性的样品系列的组成的实例。在这种情况下,脂质储备溶液的浓度为40mM,而洗涤剂储备溶液的浓度为10mM
      表1.样品系列示例:用洗涤剂提取脂质


    2. 然后将悬浮液涡旋并在给定温度下使用水浴孵育至少30分钟。
    3. 孵育后,将样品在20,800×g和1℃下离心5分钟。提取的脂质定义为存在于保留在上清液中的小扰动剂/脂质组装体中的脂质,而剩余的MLV(可能具有结合的扰动剂)团粒。对照样品(无扰动剂)的离心通常显示在颗粒中存在超过95%的脂质。
      注意:可以选择另一个温度用于离心;对照样品(无扰动剂)提供了通过离心从所剩余的MLV中分离提取的脂质的效率的定量描述。
    4. 使用注射器分离上清液
    5. 将沉淀重悬于500μlMOPS缓冲液中用于分析
  3. 脂质分析
    进行两个平行分析以确定脂质提取的程度。首先,通过Bartlett磷测定法(Bartlett,1958)测定上清液和沉淀中的磷脂含量。第二,对上清液和沉淀进行LC-MS分析
  4. Bartlett磷测定
    1. 将含有8至70nmol磷脂的脂质样品的等分试样转移到玻璃试管中。通常,使用20μl的上清液和20μl的重悬沉淀物
    2. 在玻璃试管中制备含有0(空白)和3.5mM磷酸盐的标准样品,从KH sub 2 PO 4储备溶液(3.5mM,在Milli-Q水; 47.63mg,在100ml中)。通常,制备具有3.5,2.0,1.5,1.0,0.4mM KH 2 PO 4 sub的样品。
    3. 将每份标准样品的20μl等分试样转移到玻璃试管中
    4. 向每个管中加入120μl浓硫酸。然后涡旋样品。
    5. 向每个管中加入20μlH 2 O 2 sub(30%)。然后涡旋样品。
    6. 将管全部一起加热至200℃10分钟。在加热期间,大理石放置在管的顶部以防止潜在的蒸发
    7. 将样品放在工作台上并使其冷却至室温
    8. 向每个试管中加入1340μlMilli-Q水。然后涡旋样品。
    9. 向每个试管中加入40μlNa 2 S 2 Sub 2 O 5溶液(在Milli-Q水中为526mM; 100mg在1ml中) 。然后涡旋样品。
    10. 将管一起加热至〜90℃,正好5分钟。
    11. 将样品放在工作台上并使其冷却至室温
    12. 向每个试管中加入400μl的(NH 4)6 Sub 6 Mo 7 O 24 S 4 4H 2 (16.2mM,在Milli-Q水中; 200mg,在10ml中)。然后涡旋样品。
    13. 向每个管中加入40μl抗坏血酸(568mM,在Milli-Q水中; 100mg,在1ml中,该溶液是光敏的,并且在其使用前必须保持在黑暗中)。然后涡旋样品。
    14. 将管一起加热至〜90℃,恰好10分钟。将出现蓝色。
    15. 将样品放在工作台上并使其冷却至室温
    16. 在1小时内读取820nm处的吸光度,并用于磷脂测定(图1)

      图1. Bartlett分析的典型校准曲线

  5. LC-MS分析
    通过LC-MS分析测定上清液和沉淀的脂质组成。通常注射每个样品的32μl等分试样,并对每种条件进行三次重复。样品在保持在50℃的YMC二醇柱(4.6×150mm,5μm粒径)(Agilent Technologies)上洗脱。使用乙腈/乙酸铵水溶液(100mM)(85/15,v/v)以0.6ml/min洗脱磷脂7分钟。 ESI源用于正离子化模式。在250℃和12L/min下使用氮作为干燥气体。雾化气体也是氮气,保持在241kPa。
    1. 将2μl等份的上清液和重悬浮的沉淀物在1ml H 2 O/MeOH(1/9,v/v)溶液中稀释。
    2. 注射2μl等分试样用于LC/MS分析

数据分析

  1. Bartlett测定
    提取的磷脂的分数f由下式提供:

  2. LC/MS分析
    使用以下来确定每种脂质的提取程度:
    提取%= As /(As + Ap)×100%
    在哪里,
    As和Ap分别是来自上清液和沉淀物分析的脂质峰面积。提取的脂质特异性通过比较每种脂质计算的提取%来获得。在不存在特异性的情况下,不同脂质的提取%应该相同。一种脂质相对于另一种脂质的提取%因子定义为其特定的提取
    实施例:表2显示了洗涤剂诱导的磷脂酰胆碱(PC)/磷脂酰乙醇胺(PE)膜的脂质选择性的结果的实例。

    表2.样品系列的实施例:通过洗涤剂提取脂质的选择性脂质浓度恒定(1.5mM),洗涤剂浓度变化。

笔记

  1. 对于Bartlett测定,独立三次重复的重现性通常为8%,优于10%。
  2. 对于LC/MS分析,3次进样之间的重现性通常优于1%,对于独立的三次进样,其优于5%。
  3. 对于0.75和4mM之间的洗涤剂浓度,萃取是PC特异性的,因为相对于PE萃取更多的PC。当MLV完整或完全破坏时,不能表达脂质提取特异性
  4. LC/MS和Bartlett测定法之间的总磷脂提取的一致性反映了测定的准确性。

致谢

这项工作得到加拿大自然科学和工程研究理事会以及魁北克自然和技术通过其战略集群计划的支持。该方案改编自以前的工作(Therrien等人,2013; 2016)。

参考文献

  1. Bartlett,GR(1958)。  Phosphorous assay in column chromatography。 J Biol Chem 234:466-468。
  2. Bechinger,B。(2014)。  SMART模型:软膜在抗微生物肽的存在下适应和反应,也瞬时。

  3. Schaefer,EJ,Anthanont,P.和Asztalos,BF(2014)。  高密度脂蛋白代谢,组成,功能和缺乏 Curr Opin Lipidol 25:194-199。 />
  4. Strömstedt,AA,Ringstad,L.,Schmidtchen,A.和Malmsten,M。(2010)。  两亲性肽和磷脂膜之间的相互作用。 Curr Opin Colloids Interface Sci 15:467-478。
  5. Therrien,A.,Fournier,A.和Lafleur,M.(2016)。  蜂毒素的阳离子C末端片段对膜破裂的作用。物理化学B 120(17):3993-4002。
  6. Therrien,A.,Manjunath,P.和Lafleur,M。(2013)。  用精子BSP1的牛粘合剂进行脂质提取的化学和物理要求。生物化学生物物理学(Biochim Biophys Acta)1828(2):543-551。
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引用:Lafleur, M. and Therrien, A. (2016). Determining Efficiency and Selectivity of Lipid Extraction by Perturbing Agents from Model Membranes. Bio-protocol 6(22): e2016. DOI: 10.21769/BioProtoc.2016.
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