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Extraction and Profiling of Plant Polar Glycerol Lipids
植物极性甘油脂质的提取和分析   

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Abstract

This protocol describes a method to extract total polar glycerol lipids from plant materials, followed by mass spectrometry profiling. Different glycerol lipid classes can be distinguished by their head-groups, which can be profiled automatically and quantitatively by a triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode with an autosampler. Comparing with other established methods, such as thin layer chromatography (TLC) separation followed by Gas spectrometry (GC) analysis, this method requires little effort in sample preparation and separation, while the resolution is not limited to general lipid classes but at side chain level. This method was described and used successfully to profile plant lipids changes under freezing stress in Welti et al. (2002).

Keywords: Mass spectrometry(质谱法), Lipidomics(脂类组学), Phospholipids(磷脂), Glycolipids(糖脂)

Materials and Reagents

  1. PYREXTM screw cap culture tubes with PTFE lined phenolic caps (Thermo Fisher Scientific, catalog number: 14-933D )
  2. Disposable glass pipettes (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 13-678-20C )
  3. Autosampler vials (Sigma-Aldrich, catalog number: 29133-U ) with pre-cut cap (Sigma-Aldrich, catalog number: 29494-U )
  4. TLC Silica gel 60 plate (Merck)
  5. 1 ml syringe
  6. Injection loop
  7. 4-6 weeks old Arabidopsis rosette leaves
    Note: Other plant materials can be used, additional precautions may be needed (see Notes).
  8. Isopropanol (Thermo Fisher Scientific, catalog number: A416 )
  9. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  10. Chloroform (Thermo Fisher Scientific, catalog number: C607 )
  11. Methanol (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: A456 )
  12. Acetic acid
  13. Iodine
  14. Primuline
  15. Acetone
  16. Nitrogen gas
  17. Butylated hydroxytoluene (BHT) (Sigma-Aldrich, catalog number: B1378 )
  18. 7.5 M ammonium acetate solution (NH4Ac) (Sigma-Aldrich, catalog number: A2706 )
  19. Phospholipid (PL) standard [kindly provided by Dr. Ruth Welti, (Kansas State University)]
    Note: An example PL standard list is shown in Table 1.
  20. Monogalactosyldiacylglycerol (MGDG), hydrogenated (Matreya, catalog number: 1058 )
  21. Digalactosyldiacylglycerol (DGDG), hydrogenated (Matreya, catalog number: 1059 )
    Note: The actual composition of MGDG and DGDG standards need to be determined experimentally, such as transmethylation followed by GC analysis (Politz et al., 2013).
  22. Isopropanol solution with BHT (see Recipes)
  23. Lipid extraction solution (see Recipes)
  24. KCl solution (see Recipes)
  25. Solution B (see Recipes)
  26. Galactolipid (GL) standard (see Recipes)
  27. Phospholipid (PL) standard (see Recipes)

Equipment

  1. Water bath (Thermo Fisher Scientific, model: Isotemp2340 )
  2. Water bath shaker (Labnet International, model: SWB5050 )
  3. Nitrogen evaporator N-EVAP (Organomation, model: 111 )
  4. Fume hood
  5. Ultraviolet light (360 nm)
  6. Oven (Thermo Fisher Scientific, Fisher ScientificTM, model: 13-247-650G )
  7. API 4000 LC-MS/MS system (AB SCIEX) with TurboIonspray probe and HTC PAL autosampler (LEAP Technologies)

    Table 1. Example of PL standard list

Software

  1. Analyst 1.5.1 (AB SCIEX)

Procedure

  1. Total lipid extraction (Figure 1)
    1. Aliquot 3 ml preheated isopropanol (75 °C with 0.01% BHT) into glass tubes (50 ml, 25 x 150 mm tubes with Teflon-lined screw caps are recommended).
    2. Fresh Arabidopsis leaves were harvested and immediately soaked in hot isopropanol for at least 15 min in 75 °C water bath to inactivate phospholipase D. To achieve higher reliability and repeatability, 5-30 mg delipidated “dry weight” as measured in step A10 is recommended (~2-8 fully developed leaves to start with). Samples were cooled to room temperature after incubation. Isopropanol treated samples are stable at room temperature within a day.
    3. Add 1.5 ml chloroform and 0.6 ml water to cooled samples, mix by vortexing. Incubate samples in water bath shaker at room temperature, 50 rpm for 1 h.
    4. Transfer extracted lipids (disregard potential phase separation) with a glass Pasteur pipettes to another glass collection tube.
    5. Add 4 ml lipid extraction solution to leaf samples and incubate in 25 °C water bath shaker for 30 min. Transfer extracts to the same collection tube.
    6. Repeat step A5 to extract samples with lipid extraction solution until all leaf samples become white. It is recommended to extract each sample the same number of times. 5 extractions (including isopropanol in step A2) are usually needed. Old leaves are harder to be extracted completely, longer incubation time in steps A2, A3 and A5 and more extraction steps may help.
    7. Add 1 ml KCl solution to the combined extract (~20 ml), mix thoroughly by vortex, centrifuge at ~200 x g for 10 min, discard upper phase carefully.
    8. Add 2 ml water, mix by vortex, centrifuge at ~200 x g for 10 min, discard upper phase carefully.
    9. Dry lipid samples completely in the nitrogen evaporator, mild heating (<50 °C) may be used to accelerate the process. Dried lipids can be stored at -80 °C with cap tightly closed. An aliquot of lipids can be resolved by thin layer chromatography (TLC) for quality check (Figure 2). Briefly, total lipids corresponding to ~0.2-1 mg DW were spotted on the TLC Silica gel 60 plate and resolved by chloroform:methanol:acetic acid:H2O = 85:15:12.5:3.5. The plate was left dry completely in the fume hood before staining by iodine vapor and examining by eye or 0.01% primuline in acetone/H2O (60:40; v/v) and examining the plates under ultraviolet (360 nm) light.
    10. Dry extracted leaves in oven at 105 °C overnight. Weigh to get “dry weight”.

  2. Lipid profiling by ESI-MS/MS and data processing
    1. Reconstitute dried lipid samples (step A9) in chloroform, cap tightly before use to avoid evaporation. For easy sample preparation, lipid samples were diluted to the same concentration according to dry weight. We usually use 250 µl chloroform per mg dry weight.
    2. Each sample contains 840 µl solution B, 5 µl PL standard, 3 µl GL standard and lipid sample corresponding to 0.1-0.2 mg dry weight (25-50 µl), add chloroform to 1,200 µl in a 2 ml snap seal autosampler vial, cap with pre-cut vial caps.
    3. For every 10 lipid samples, only one “internal standard (IS)” vial is used for machine correction/contamination control. Chloroform is used in place of lipid samples in this vial.
    4. Load the autosampler in the following format: IS1, sample1-10, IS2, sample11-20 and so on. “Blank” samples are recommended between different genotypes, which contains only solution B and chloroform.
    5. Profile lipid samples by ESI-MS/MS. Detailed information can be found in Welti et al. (2002). The parameters we used on API 4000 are listed in section C. Optimization is needed on different setups, such that fewer or more parameters can be adjusted on older or newer machines. A representative phosphatidylethanolamine (PE) profiling result is attached (Figure 3).
    6. Data were extracted by Analyst 1.5.1 with necessary corrections, including background subtraction and smooth. For each lipid class, multiple (usually 2) novel lipid species were used as internal standards (Table 1), linear interpolation according to molecular weight of each lipid species was used for internal standard peak area normalization. The amount of each lipid species was calculated according to sample peak area compared with normalized internal standard peak area. Five or more biological repeats are recommended, Student’s t-test is used for statistical analysis.
    7. Lipid data may be presented as absolute amount (nmol/mg “dry weight”, recommended) or percentage of total lipids (PL and GL).

  3. Lipid profiling parameters on API 4000
    API 4000 LC-MS/MS system equipped with TurboIonSpray probe and HTC PAL autosampler was used for lipid profiling. The source temperature was 100 °C, interface heater was on, IonSpray Voltage was set to 5.5 kV, nitrogen gas was used for both source and curtain gas. The scan resolution was set to 0.1Da. The samples were introduced using the autosampler with 1 ml syringe and injection loop for full lipid profiling. The samples were presented to the TurboIonSpray probe by direct infusion at 0.03 ml/min. The scanning parameters for each lipid class were listed in Table 2.

    Table 2. Scanning parameters

Representative data



Figure 1. Illustration of lipid extraction workflow. 1,000 rpm (~200 x g).


Figure 2. TLC separation of phospholipids. Lipids extracted in section A can be resolved on a TLC plate. The mobile phase used here is chloroform:methanol:acetic acid:H2O = 85:15:12.5:3.5 (v/v).


Figure 3. Representative MS data. A. Different lipid classes were profiled in tandem periods. Period 1 is for control purpose and not used in calculation. B. PE profile in IS sample. C. PE profile in plant sample. D. Enlarged view of plant PE species.

Notes

  1. This Method can be easily adapted to roots, flowers, stems and whole siliques.
    1. For Arabidopsis seed samples, the reproducibility of “dry weight” is highly dependent on seed stage and loss of samples during transferring lipid extracts. In this case, the percentage normalization is recommended.
    2. For materials that are hard to extract completely, tissue disruption in liquid nitrogen before extraction is recommended. In this case, a centrifugation step (e.g., ~200 x g, 10 min) is needed each time before transferring lipid extracts.

Recipes

  1. Isopropanol solution with BHT
    0.01% (w/v) BHT (~0.5 mM)
    Preheat at 75 °C
    Note: stable at room temperature
  2. Lipid extraction solution
    Chloroform:methanol = 2:1 (v/v), with 0.01% BHT
    Note: stable if capped tightly at room temperature
  3. KCl solution
    1 M KCl
    Note: stable at room temperature
  4. Solution B
    Methanol:NH4Ac (300 mM) = 20:1(v/v)
    Note: stable at room temperature
  5. Galactolipid (GL) standard
    Note: store at -80 °C, thaw and mix thoroughly before use
    Diluted in chloroform:methanol:H2O = 35:65:8 [(v/v), or close to]
    MGDG (~0.5 nmol/µl)
    DGDG (~0.25 nmol/µl)
  6. Phospholipid (PL) standard
    Note: store at -80 °C, thaw and mix thoroughly before use
    Diluted in chloroform:methanol:H2O = 35:65:8 [(v/v), or close to]
    See Table 1 for an example of PL standard content

Acknowledgments

This protocol was adapted from Welti et al. (2002). Dr. Welti also provided help in lipid standards, data processing and analysis. Work was supported by the U.S. Department of Energy (DOE) Grant DE-AR0000202 and by the National Science Foundation Grants MCB-1412901 and DBI-1427621.

References

  1. Politz, M., Lennen, R. and Pfleger, B. (2013). Quantification of bacterial fatty acids by extraction and methylation. Bio-protocol 3(21): e950.
  2. Welti, R., Li, W., Li, M., Sang, Y., Biesiada, H., Zhou, H. E., Rajashekar, C. B., Williams, T. D. and Wang, X. (2002). Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. J Biol Chem 277(35): 31994-32002.

简介

该方案描述了从植物材料提取总极性甘油脂质的方法,随后是质谱分析。 不同的甘油脂类可以通过其头基来区分,其可以通过使用自动进样器的多重反应监测(MRM)模式中的三重四极杆质谱自动和定量地进行概况分析。 与其他已建立的方法,例如薄层色谱(TLC)分离,然后气体光谱(GC)分析相比,该方法在样品制备和分离中几乎不需要付出努力,而分辨率不限于一般脂质类别,而是侧链水平 。 在Welti等人(2002)中描述并且成功地使用该方法来分析在冰冻胁迫下植物脂质的变化。

关键字:质谱法, 脂类组学, 磷脂, 糖脂

材料和试剂

  1. 带有PTFE内衬酚醛帽的PYREX TM螺旋盖培养管(Thermo Fisher Scientific,目录号:14-933D)
  2. 一次性玻璃移液管(Thermo Fisher Scientific,Fisher Scientific ,目录号:13-678-20C)
  3. 具有预切帽的自动进样器小瓶(Sigma-Aldrich,目录号:29133-U)(Sigma-Aldrich,目录号:29494-U)
  4. TLC硅胶60板(Merck)
  5. 1 ml注射器
  6. 注入回路
  7. 4-6周龄的拟南芥玫瑰花叶
    注意:可以使用其他植物材料,可能需要额外的预防措施(见注释)。
  8. 异丙醇(Thermo Fisher Scientific,目录号:A416)
  9. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  10. 氯仿(Thermo Fisher Scientific,目录号:C607)
  11. 甲醇(Thermo Fisher Scientific,Fisher Scientific ,目录号:A456)
  12. 乙酸

  13. 普Prim素
  14. 丙酮
  15. 氮气
  16. 丁基化羟基甲苯(BHT)(Sigma-Aldrich,目录号:B1378)
  17. 7.5M乙酸铵溶液(NH 4 Ac)(Sigma-Aldrich,目录号:A2706)
  18. 磷脂(PL)标准[由Dr. Ruth Welti,(堪萨斯州立大学)提供]
    注意:示例PL标准列表如表1所示。
  19. 单半乳糖基二酰基甘油(MGDG),氢化(Matreya,目录号:1058)
  20. 二半乳糖基二酰基甘油(DGDG),氢化(Matreya,目录号:1059)
    注意:MGDG和DGDG标准物的实际组成需要通过实验确定,例如甲基化,然后进行GC分析(Politz et al。,2013)。
  21. BHT的异丙醇溶液(参见配方)
  22. 脂质提取溶液(参见配方)
  23. KCl溶液(见配方)
  24. 解决方案B(参见配方)
  25. Galactolipid(GL)标准(见配方)
  26. 磷脂(PL)标准(参见配方)

设备

  1. 水浴(Thermo Fisher Scientific,型号:Isotemp2340)
  2. 水浴摇动器(Labnet International,型号:SWB5050)
  3. 氮气蒸发器N-EVAP(Organomation,型号:111)
  4. 通风橱
  5. 紫外光(360nm)
  6. Oven(Thermo Fisher Scientific,Fisher Scientific ,型号:13-247-650G)
  7. 带有TurboIonspray探针和HTC PAL自动进样器(LEAP Technologies)的API 4000 LC-MS/MS系统(AB SCIEX)

    表1. PL标准列表示例

软件

  1. 分析者1.5.1(AB SCIEX)

程序

  1. 总脂质提取(图1)
    1. 推荐将3 ml预热的异丙醇(75°C,0.01%BHT)放入玻璃管(50 ml,25 x 150 mm,配有Teflon内衬螺旋盖)中。
    2. 收获新鲜的拟南芥叶,并立即在75℃水浴中在热异丙醇中浸泡至少15分钟以灭活磷脂酶D.为了实现更高的可靠性和重复性,5-30mg脱脂"干重"推荐在步骤A10中测量(?2-8完全发育叶开始)。温育后将样品冷却至室温。异丙醇处理的样品在室温下在一天内是稳定的
    3. 向冷却的样品中加入1.5ml氯仿和0.6ml水,通过涡旋混合。将样品在室温,50rpm的水浴摇动器中孵育1小时
    4. 用玻璃巴斯德移液管将提取的脂质(忽略电位相分离)转移到另一个玻璃收集管
    5. 加入4毫升脂质提取液到叶片样品,并在25℃水浴摇床中孵育30分钟。将提取物转移到同一收集管。
    6. 重复步骤A5,用脂质提取溶液提取样品,直到所有叶样品变白。建议提取每个样品相同的次数。通常需要5次萃取(包括步骤A2中的异丙醇)。老叶子更难完全提取,更长的孵化时间在步骤A2,A3和A5和更多的提取步骤可能有帮助。
    7. 将1ml KCl溶液加入到合并的提取物(?20ml)中,通过涡旋充分混合,在?200×g离心10分钟,仔细弃去上层。
    8. 加入2ml水,涡旋混合,以?200×g离心10分钟,仔细弃去上层。
    9. 在氮气蒸发器中完全干燥脂质样品,温和加热(<50℃)可用于加速该过程。干燥的脂质可以在盖子密闭的情况下储存在-80℃。脂质的等分试样可以通过薄层色谱(TLC)拆分用于质量检查(图2)。简言之,将对应于约0.2-1mg DW的总脂质点在TLC硅胶60板上,并通过氯仿:甲醇:乙酸:H 2 O = 85:15:12.5:3.5拆分。将板在通风橱中完全干燥,然后通过碘蒸气染色并用丙酮/H 2 O(60:40; v/v)中的0.01%甘草碱检查,并检查板紫外线(360nm)光
    10. 干提取叶在105℃的烘箱中过夜。称重以获得"干重"
  2. 通过ESI-MS/MS和数据处理进行脂质谱分析
    1. 在氯仿中重构干燥的脂质样品(步骤A9),在使用前盖紧以避免蒸发。为了容易的样品制备,将脂质样品根据干重稀释至相同的浓度。我们通常使用250微升氯仿/毫克干重
    2. 每个样品包含840μl溶液B,5μlPL标准品,3μlGL标准品和脂质样品,对应于0.1-0.2mg干重(25-50μl),在2ml快速封口自动进样器小瓶中加入氯仿至1,200μl,带预切小瓶盖。
    3. 对于每10个脂质样品,一个"内标(IS)"仅用于机器校正/污染控制。使用氯仿代替该小瓶中的脂质样品
    4. 以下列格式加载自动进样器:IS1,sample1-10,IS2,sample11-20等。在不同基因型之间推荐"空白"样品,其仅含有溶液B和氯仿
    5. 通过ESI-MS/MS分析脂质样品。详细信息可以在Welti等人的(2002)中找到。我们在API 4000上使用的参数在第C节中列出。在不同的设置上需要优化,以便在较旧或较新的机器上调整更少或更多的参数。附着代表性的磷脂酰乙醇胺(PE)分析结果(图3)
    6. 数据由Analyst 1.5.1提取,必要的校正,包括背景扣除和平滑。对于每种脂类,使用多种(通常为2种)新型脂质作为内标(表1),根据每种脂质的分子量的线性内插用于内标峰面积归一化。根据与标准化内标峰面积比较的样品峰面积计算每种脂质种类的量。推荐使用五个或更多的生物学重复,使用学生t检验进行统计分析
    7. 脂质数据可以表示为绝对量(nmol/mg"干重",推荐)或总脂质(PL和GL)的百分比。

  3. API 4000上的脂质分析参数
    配备有TurboIonSpray探针和HTC PAL自动进样器的API 4000 LC-MS/MS系统用于脂质分析。源温度为100℃,界面加热器开启,离子喷雾电压设定为5.5kV,氮气用于源气和帘幕气。扫描分辨率设置为0.1Da。使用具有1ml注射器和注射环的自动进样器引入样品用于全脂质分析。通过以0.03ml/min直接注入将样品提供给TurboIonSpray探针。每种类脂的扫描参数列于表2中
    表2.扫描参数

代表数据



图1.脂质提取工作流程示意图。 1,000 rpm(?200 x g )。


图2.磷脂的TLC分离。 在A部分提取的脂质可以在TLC板上分离。这里使用的流动相是氯仿:甲醇:乙酸:H 2 O = 85:15:12.5:3.5(v/v)。

图3.代表性MS数据。A.在不同时期分析不同的脂质类型。期间1用于控制目的,不用于计算。 B.IS样品中的PE曲线。 C.植物样品中的PE谱。 D.植物PE物种的放大视图。

笔记

  1. 这种方法可以很容易适应根,花,茎和整个长角果。
    1. 对于拟南芥种子样品,"干重"的可重复性高度依赖于转移脂质提取物期间的种子阶段和样品损失。在这种情况下,建议使用百分比归一化。
    2. 对于难以完全提取的材料,建议在提取前在液氮中进行组织破坏。在这种情况下,每次在转移脂质提取物之前需要离心步骤(例如,,?200×g×10分钟,10分钟)。

食谱

  1. 用BHT的异丙醇溶液
    0.01%(w/v)BHT(?0.5mM) 在75°C预热
    注意:在室温下稳定
  2. 脂质提取液
    氯仿:甲醇= 2:1(v/v),用0.01%BHT洗脱 注意:如果在室温下盖紧,则稳定
  3. KCl溶液
    1 M KCl
    注意:在室温下稳定
  4. 解决方案B
    甲醇:NH 4 Ac(300mM)= 20:1(v/v) 注意:在室温下稳定
  5. Galactolipid(GL)标准
    注意:存放于-80°C,使用前请彻底融化并混匀
    在氯仿:甲醇:H 2 O = 35:65:8 [(v/v)或接近]中稀释。
    MGDG(?0.5nmol /μl)
    DGDG(?0.25nmol /μl)
  6. 磷脂(PL)标准
    注意:存放于-80°C,使用前请彻底融化并混匀
    在氯仿:甲醇:H 2 O = 35:65:8 [(v/v)或接近]中稀释。
    PL标准内容的示例参见表1

致谢

该方案改编自Welti等人(2002)。 Welti博士还提供脂质标准,数据处理和分析方面的帮助。工作由美国能源部(DOE)Grant DE-AR0000202和国家科学基金会拨款MCB- 1412901 和DBI- 1427621

参考文献

  1. Politz,M.,Lennen,R。和Pfleger,B。(2013)。  分析膜脂质在植物胁迫反应中的作用。磷脂酶Dα在拟南芥中的冷冻诱导的脂质变化中的作用。

    J Biol Chem 277(35):31994-32002。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, Y. and Wang, X. (2016). Extraction and Profiling of Plant Polar Glycerol Lipids. Bio-protocol 6(12): e1849. DOI: 10.21769/BioProtoc.1849.
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