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Positional Analysis of Fatty Acids in Phospholipids by PLA2 Treatment
采用PLA2处理法对磷脂中的脂肪酸进行位置分析   

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参见作者原研究论文

本实验方案简略版
The Plant Cell
May 2015

Abstract

Plant phospholipids can be produced in the endoplasmic reticulum or plastids. Lipids from different sources can be distinguished by the fatty acid profile, in terms of the preferred fatty acid species esterified to the sn-1 or sn-2 position of the glycerol backbone (Ohlrogge and Browse, 1995). This protocol is used to determine the fatty acid profile in total plant phospholipids by the treatment of sn-2 specific phospholipase A2 (PLA2).

Keywords: Lipidomics (脂类组学), Phospholipase A2 (磷脂酶A2), Prokaryotic pathway (核途径), Eukaryotic pathway (真核生物的途径)

Materials and Reagents

  1. Glass tubes [PYREXTM screw cap culture tubes with PTFE lined phenolic caps (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 14-932H )]
  2. Pipette 100-1,000 µl (Eppendorf Research 2100)
  3. AxygenTM 1,000 µl universal pipette tips (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 14-222-690 )
  4. Total plant lipids
    Note: Extracted by the method described in the companion lipid extraction and profiling protocol by the same authors (Liu and Wang, 2016) and the original publication (Welti et al., 2002)
  5. Chloroform (Thermo Fisher Scientific, catalog number: C607 )
  6. PLA2 from honey bee venom (Sigma-Aldrich, catalog number: P9279 )
    Note: Dissolved in reaction buffer to 1 unit/µl before use.
  7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  8. Nitrogen gas
  9. HEPES (Sigma-Aldrich, catalog number: H3375 )
  10. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C8106 )
  11. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  12. Methanol (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: A456 )
  13. Reaction buffer (see Recipes)
  14. NaCl solution (see Recipes)
  15. Extraction solution (see Recipes)

Equipment

  1. Nitrogen evaporator N-EVAP (Meyer Organomation, model: 111 )
  2. Ultrasonic processor with probe, work at 30% power (GEX 130PB) or similar equipment [e.g., ultrasonic processor, (Cole-parmer, model: EW-04714-51 )]
  3. Incubator (Thermo Fisher Scientific, model: Isotemp 537D ) or similar equipment [e.g., microbiological incubators (Thermo Fisher Scientific, IsotempTM, catalog number: 15-103-0513 )]

Procedure

  1. Total plant lipids were reconstituted by chloroform and the amount of lipids corresponding to 0.2 mg dry weight were aliquoted in glass tubes. Same amount of untreated lipid samples will be used as controls in step 9.
  2. Lipids were dried in the nitrogen evaporator thoroughly (>30 min), mild heating (<50 °C) may be used to accelerate the process.
  3. Dried lipids were hydrated in 500 µl reaction buffer at room temperature for >10 min, followed by sonication (30% power, on ice) to disperse the lipids uniformly.
  4. 10 units (10 µl) of PLA2 were added to the reaction, mixed by vortex. The reaction mixtures were incubated at 37 °C for 2 h to completely digest phospholipids.
  5. 500 µl of 500 mM NaCl was added to the mixture to facilitate phase separation. 2 ml extraction solution was added and vortexed to terminate the reaction and extract lipids.
  6. Centrifuge at ~200 x g for 10 min. Transfer the bottom (organic) phase to another glass tube.
  7. Add 1 ml chloroform and repeat step 6, twice for complete extraction.
  8. Dry extracted lipids under nitrogen gas, reconstitute the lipids in 50 µl chloroform.
  9. Use total lipids corresponding to 0.2 mg dry weight as control, profile both digested and undigested lipids in mass spectrometry as described in the companion lipid extraction and profiling protocol by the same authors (Liu and Wang, 2016). Three or more biological repeats are recommended and Student’s t-test is used for statistical analysis. Compare regular lipids in undigested samples and lysolipids in digested samples to determine position/composition of fatty acid in different lipid classes. For more quantitative results and more phospholipids classes besides phosphatidylcholine (PC) and phosphatidylethanolamine (PE), lipid samples can first be resolved by thin layer chromatography (TLC) (Refer to the method in Liu et al., 2015) and each lipid/lysolipid spot can be quantified by transmethylation and GC analysis (Politz et al., 2013).

Representative data

Representative data from Liu et al., 2015 (Figure 1). Total lipids were digested by PLA2, followed by analysis of PC and LysoPC by mass spectrometry. The increase of LPC16 in digested sample and increase of PC34 in undigested samples in RNAi lines compared with WT are similar, suggesting the increased PC34 in RNAi lines have C16 fatty acid esterified to the sn-1 position, which originates from the eukaryotic pathway.


Figure 1. Analysis of fatty acyl chain position/composition in phosphatidylcholine (PC). A. Content of LysoPC with C16 and C18 fatty acids after PLA2 treatments of lipids from two AAPT RNAi lines and WT plants. B. Content of PC with C34 and C36 fatty acids without PLA2 treatments of lipids from two AAPT RNAi lines and WT plants. * and ** mark differences between WT and mutant at P < 0.05 and at P < 0.01, respectively based on Student’s t-test. Values are means ± SD (n = 3).

Recipes

  1. Reaction buffer
    100 mM HEPES, pH 7.4
    10 mM CaCl2
    10 mM MgCl2
    Note: Stable in room temperature.
  2. NaCl solution
    500 mM NaCl
    Note: Stable in room temperature.
  3. Extraction solution
    66.7% (v/v) Chloroform
    33.3% (v/v) Methanol
    Note: made before use

Acknowledgments

This protocol is adapted from the work of Williams et al., 1995. Work was supported by the National Institute of Food and Agriculture Grant 2016-67013-24429 and the U.S. Department of Energy (DOE) Grant DE-AR0000202.

References

  1. Liu, Y. and Wang, X. (2016). Extraction and profiling of plant polar glycerol lipids. Bio-protocol 6(12): e1849.
  2. Liu, Y., Wang, G. and Wang, X. (2015). Role of aminoalcoholphosphotransferases 1 and 2 in phospholipid homeostasis in Arabidopsis. Plant Cell 27(5): 1512-1528.
  3. Ohlrogge, J. and Browse, J. (1995). Lipid biosynthesis. Plant Cell 7(7): 957-970.
  4. Politz, M., Lennen, R. and Pfleger, B. (2013). Quantification of bacterial fatty acids by extraction and methylation. Bio-protocol 3(21): e950.
  5. Williams, J. P., Khan, M. U. and Wong, D. (1995). A simple technique for the analysis of positional distribution of fatty acids on di- and triacylglycerols using lipase and phospholipase A2. J Lipid Res 36(6): 1407-1412.
  6. 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

植物磷脂可以在内质网或质体中产生。 来自不同来源的脂质可以通过脂肪酸谱来区分,根据酯化为甘油主链的sn-1或em-sn-2位置的优选脂肪酸种类 (Ohlrogge和Browse,1995)。 该方案用于通过处理sn-2特异性磷脂酶A 2(PLA 2)来确定总植物磷脂中的脂肪酸谱。 。

关键字:脂类组学, 磷脂酶A2, 核途径, 真核生物的途径

材料和试剂

  1. 玻璃管[具有PTFE内衬酚醛帽的PYREX 螺旋盖培养管(Thermo Fisher Scientific,Fisher Scientific ,目录号:14-932H)]
  2. 移液管100-1000μl(Eppendorf Research 2100)
  3. Axygen TM 1,000μl通用移液管吸头(Thermo Fisher Scientific,Fisher Scientific TM ,目录号:14-222-690)
  4. 总植物脂质
    注意:由相同作者(Liu和Wang,2016)和原始出版物(Welti等人,2002)在伴随脂质提取和分析方案中描述的方法提取。
  5. 氯仿(Thermo Fisher Scientific,目录号:C607)
  6. 来自蜂蜜蜂毒液(Sigma-Aldrich,目录号:P9279)的PLA 2
    注意:在使用前溶解在反应缓冲液中1单位/μl。
  7. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  8. 氮气
  9. HEPES(Sigma-Aldrich,目录号:H3375)
  10. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C8106)
  11. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)
  12. 甲醇(Thermo Fisher Scientific,Fisher Scientific ,目录号:A456)
  13. 反应缓冲液(参见配方)
  14. NaCl溶液(见配方)
  15. 提取溶液(见配方)

设备

  1. 氮气蒸发器N-EVAP(Meyer Organomation,型号:111)
  2. 具有以30%功率工作的探头的超声处理器(GEX 130PB)或类似设备[例如,超声波处理器(Cole-parmer,型号:EW-04714-51)]
  3. 孵育器(Thermo Fisher Scientific,型号:Isotemp 537D)或类似设备[例如,微生物培养箱(Thermo Fisher Scientific,Isotemp TM,目录号:15-103-0513 )]

程序

  1. 通过氯仿重建总植物脂质,并将对应于0.2mg干重的脂质的量等分在玻璃管中。在步骤9中将使用相同量的未处理的脂质样品作为对照
  2. 将脂质在氮气蒸发器中彻底干燥(> 30分钟),可以使用温和加热(<50℃)以加速该过程。
  3. 干燥的脂质在500μl反应缓冲液中在室温下水合≥10分钟,随后超声(30%功率,在冰上)以均匀地分散脂质。
  4. 将10单位(10μl)的PLA 2加入到反应中,通过涡旋混合。将反应混合物在37℃下孵育2小时以完全消化磷脂
  5. 向混合物中加入500μl500mM NaCl以促进相分离。加入2ml提取溶液并涡旋以终止反应并提取脂质
  6. 以?200×g离心10分钟。将底部(有机)相转移到另一个玻璃管
  7. 加入1ml氯仿,重复步骤6,两次完全萃取
  8. 在氮气下干燥提取的脂质,在50μl氯仿中重构脂质
  9. 使用对应于0.2mg干重的总脂质作为对照,在质谱中描述消化和未消化脂质,如同一作者的伴随脂质提取和分析方案中所述(Liu和Wang,2016)。推荐三个或更多个生物学重复,并且使用Student's t检验进行统计分析。比较未消化样品中的常规脂质和消化样品中的溶血脂质,以确定不同脂质类别中脂肪酸的位置/组成。对于除了磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)之外的更多的定量结果和更多的磷脂类,脂质样品可以首先通过薄层色谱法(TLC)拆分(参见Liu等人的 2015),并且每个脂质/溶血脂点可以通过转甲基化和GC分析来定量(Politz等人,2013)。

代表数据

来自Liu等人的代表性数据,2015(图1)。总脂质通过PLA 2消化,然后通过质谱法分析PC和LysoPC。与野生型相比,消化样品中LPC16的增加和RNAi品系中未消化样品的PC34的增加是相似的,表明RNAi品系中增加的PC34具有酯化到sn-1位的C16脂肪酸源于真核生物途径

图1.磷脂酰胆碱(PC)中脂肪酰基链位置/组成的分析。 A。在来自两个AAPT TM RNAi系和WT植物的脂质的PLA 2处理后,具有C16和C18脂肪酸的LysoPC的含量。 B.来自两个AAPT RNAi系和WT植物的脂质的具有C34和C36脂肪酸而不进行PLA 2处理的PC的含量。 *和**分别基于学生的t检验,分别在P <0.05和P <0.01时WT和突变体之间的差异。值为平均值±SD(n = 3)。

食谱

  1. 反应缓冲液
    100mM HEPES,pH7.4 10mM CaCl 2
    10mM MgCl 2/
    注意:室温稳定
  2. NaCl溶液。
    500 mM NaCl
    注意:室温稳定
  3. 提取溶液
    66.7%(v/v)氯仿
    33.3%(v/v)甲醇
    注意:使用之前

致谢

该协议改编自Williams的工作,1995。工作由国家食品和农业研究所授予日期7067013-24429和美国能源部(DOE)Grant DE支持-AR0000202。

参考文献

  1. Liu,Y。和Wang,X.(2016)。  提取和剖析植物极性甘油脂质。 生物协议 6(12):e1849。
  2. Liu,Y.,Wang,G.and Wang,X.(2015)。  氨基醇磷酸转移酶1和2在磷脂稳态中的作用拟南芥 植物细胞 27(5):1512-1528。 >
  3. Ohlrogge,J。和Browse,J.(1995)。  脂质生物合成。 植物细胞 7(7):957-970。
  4. Politz,M.,Lennen,R。和Pfleger,B。(2013)。  使用脂肪酶和磷脂酶A2分析脂肪酸在二 - 和三酰基甘油上的位置分布的简单技术。 J Lipid Res 36(6):1407-1412。
  5. Welti,R.,Li,W.,Li,M.,Sang,Y.,Biesiada,H.,Zhou,HE,Rajashekar,CB,Williams,TDand Wang,X。(2002) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12077151"target ="_ blank">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). Positional Analysis of Fatty Acids in Phospholipids by PLA2 Treatment. Bio-protocol 6(12): e1850. DOI: 10.21769/BioProtoc.1850.
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