拟南芥极地甘油脂分析薄层色谱(TLC)再加上气相色谱(GLC)

Biology
 

Summary

极性脂提取物的成分和个别甘油脂脂肪酸组成在一个简单而强大的脂质分析实验确定。为此,甘油脂是孤立的薄层色谱法和酰基组transmethylation。气相色谱定量脂肪酰基methylesters。

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Wang, Z., Benning, C. Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC). J. Vis. Exp. (49), e2518, doi:10.3791/2518 (2011).

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Abstract

生物膜从不同的细胞环境。从一个单细胞到多细胞植物和动物,甘油脂,如卵磷脂或磷脂,形成双分子膜的化学细胞及其周围环境之间的交流的边界和接口,。植物细胞与动物不同的是,有一个特殊的光合作用的细胞器,叶绿体。错综复杂的叶绿体膜系统包含独特的甘油脂,即糖脂缺乏磷:monogalactosyldiacylglycerol(MGDG),digalactosyldiacylglycerol(DGDG),sulfoquinovosyldiacylglycerol(SQDG)4 。这些脂质的作用不仅仅是结构。在光系统I和II表示甘油脂在参与光合作用8,11的晶体结构,发现这些糖脂和其他甘油脂。在磷饥饿,DGDG转移到extraplastidic细胞膜,以弥补磷脂 9,12的损失。

我们这些脂质的合成和功能知识大部分来自已经组合的遗传和生化研究拟南芥 14。在这些研究中,一个简单的程序,为极地血脂分析是必不可少的脂质突变体的筛选和分析,将详细概述。叶脂质提取物薄层色谱(TLC)分离和甘油脂是可逆沾有碘蒸气。单体脂类刮薄层板上,并转化为脂肪酰基methylesters(脂肪酸甲酯),再加上火焰离子化检测器(FID - GLC)(图1)的气相色谱分析。该方法已被证明是一个可靠的工具,突变体筛选。例如,在tgd1,2,3,4内质网到质的脂质贩运突变体发现的基础上积累的异常galactoglycerolipid:trigalactosyldiacylglycerol(TGDG)和18:3的相对量减少(碳:双债券)在膜脂的3,13,18,20脂肪酰基群体。这种方法也适用于确定使用基板 6脂类蛋白质的酶活动。

Protocol

1。脂质提取

  1. 开始收获30毫克,从4周龄琼脂上生长植物拟南芥叶片凝固介质或土壤和他们转移到1.5 mL聚丙烯反应管脂质提取。鲜叶可闪光冷冻在液氮中,并储存在-80 ° C
  2. ,氯仿和甲酸(20点10分01秒,V / V / V),每个样品中加入300μL提取溶剂甲醇组成。用力振摇5分钟(使用的油漆摇床或类似)。
  3. 加入150μL0.2 M磷酸(H 3 PO 4),1米氯化钾(KCl)和涡简要。
  4. 13000 XG在室温离心1分钟。溶解在氯仿逐步降低血脂,将会发现到薄层板。

2。薄层色谱法(TLC)15

  1. 要准备薄层板,淹没装载30秒到0.15 M硫酸铵((NH 4)2 SO 4)解决方案的地带一个20cmx20cm硅胶涂薄层板上,淹没了30秒后,干板至少2天有盖容器内。在激活过程中铵的升华留下硫酸,质子化磷脂必要从其他甘油脂分离。
  2. 在一天的实验,激活薄层色谱板在烤箱烘烤在120 ° C为2.5小时。
  3. 激活板室温冷却后,用铅笔画出一条直线从板边(1.5厘米)的色谱的起源整个板块。
  4. 在通风橱中,慢慢地提供3 + X在较低的氯仿阶段使用200μL的黄色塑料提示下慢流 N 2 20微升移液器20μL脂质提取物。为此,聚乙烯管是连接的N 2坦克的监管。保持现货直径大于1厘米的小。每个板最多可容纳10个样品(随后GLC分析计划时)。
  5. 由于脂质点通风柜完全干燥,准备发展溶剂甲苯,丙酮,水(91毫升:30毫升:7.5毫升)组成。如果周围的空气相对湿度高,可能会受到影响分离。水在这种情况下,应减少(91毫升:30毫升:7.0毫升),以达到理想的分离。
  6. 倒入80毫升,发展成一个密封室(L:高:宽= 27.0:26.5:7.0厘米/厘米/厘米)的薄层色谱溶剂与样品结束朝下放入罐板。使用钳密封罐。溶剂,将提升该板块将被分离和血脂。开发时间是在室温下约50分钟。
  7. 当溶剂的前锋已经达到了1厘米板顶部,小心取出从水箱板,并完全干燥约10分钟的通风柜。
  8. 薄层色谱法分离血脂可与碘进行定量分析或不可逆转地用硫酸或α-萘酚染色简要可逆染色。
    1. 硫酸炭化:喷以50%硫酸在水中15分钟(图2A)在通风柜和烘烤在120℃的玻璃喷雾瓶板。
    2. α-萘酚糖脂染色:在120 ° C的板喷2.4%(W / V)10%α-萘酚(V / V)硫酸,80%(V / V)乙醇和烘烤3-5直到糖脂乐队分钟被染成粉红色或紫色(图2B)。过度治疗会导致血​​脂,由于存在硫酸试剂炭化。
    3. 碘染色:在通风橱中,放入一个封闭的薄层色谱与碘晶体罐(在一个托盘上领先的碘蒸气饱和的气氛,直到血脂是可见的底部)的板。不要暴露板太长碘碘共价键修改多不饱和脂肪酸(图2C)。另外,要避免任何氧化脂质,穿插样品车道的唯一标准线应使用巴斯德吸管插入碘晶体通过二氮是对个别标准泳道吹玻璃羊毛染色。

3。脂肪酰基酸甲酯(FAME),反应16

  1. 从薄层板上,用刀片取出硅周围确定的脂斑。刮脂含二氧化硅和转让硅微粉,玻璃管与聚四氟乙烯(PTFE)内衬聚四氟乙烯螺丝帽使用一个漏斗。
  2. 添加无水甲醇1毫升1N盐酸(盐酸)每个样品由玻璃吸管。
  3. 加入100微升50微克毫升-1十五烷酸(15:0)作为内部使用200μL200μL的黄色塑料尖吸管标准每个样品200微升移液器。 pentadecenoic甲醇盐酸酸作为控制,保持管。紧紧关闭的玻璃管与聚四氟乙烯内衬的盖子。
  4. 孵育GL屁股管在80℃水浴25分钟。管需要密封,使溶剂不蒸发。
  5. 管冷却下来后,加入1毫升0.9%氯化钠1 mL正己烷和涡大力。在1000xg离心3分钟的样品。
  6. 在通风柜中,删除与巴斯德吸管样品的正己烷/上层,并放置到一个新的13x100毫米玻璃管。
  7. 完全未经干燥的正己烷蒸发下慢流 N 2 。
  8. 在60μL正己烷溶解,导致脂肪酰基methylesters小号。转移到自动进样瓶和盖紧紧样本。样品可存放在4 ° C的短期和-20℃的几天。

4。气相色谱(GLC)10

  1. 在开始GLC之前,确保充满氦气,氢气和气缸。
  2. 必须补充足够的正己烷溶剂水库和废物的容器必须是空的。对于脂肪酰基methylesters分离,附加到本机的DB - 23列。
  3. 到自动进样器瓶。启动绿灯ChemStation软件系统的计算机上。
  4. 入口温度设定在250 °与氦流量48.6毫升分钟-1和21.93 PSI的压力分流比为30.0:1。
  5. 最初在140 ° C烘箱温度设置为2分钟,并提升至160 ° C在25℃ 分钟 -1率。然后设定温度增加160 ° C到250 ° C时,利率为8 ° C最低-1,并保持在250 ° C为4分钟减少至140 ° C率在38 ° C 最低 - 1。一个运行大约需要21分钟。
  6. 的火焰离子化检测器温度为270 ° 30.0毫升分钟 -1,400毫升分钟 -1和30.0毫升分钟 -1氦流量的空气流速与氢气流量
  7. 输入小瓶和运行序列表中的样本名。将10μL的注射器,注入2%μL样品小瓶。
  8. 当仪器已准备就绪,开始运行顺序。

5。代表性的成果:

从4周龄的拟南芥幼苗薄层色谱分离血脂不可逆转染色的例子如图2所示。硫酸染色血脂(图2A)被烧焦和褐色斑点出现。是首选α-萘酚染色,如MGDG糖脂,DGDG,SQDG与α-萘酚进行一个粉红色的紫色,而其他的极性脂类染色黄色(图2B)染色等糖脂。碘染色是可逆的,并给出血脂一个黄色超过碘蒸发(图2C)短的时间内即会消失。简要碘染​​色血脂可以受到GLC分析,虽然未染色血脂是可取的,以减少分解脂类。

如果成功,将观察到的鲜明信号,代表不同的脂肪酰基甲酯后绿灯(图3)。脂肪酸碳链较短和较少的双键酰基甲酯的保留时间较短,使用DB - 23列。脂肪酰基甲酯分析,找出与脂质成分改变的突变体是一个敏感的工具。在图4中,MGDG18:3脂肪酸的摩尔比是下降tgd4 - 1突变体比野生型的18。除以所有脂质类的痣类脂质的脂肪酰基甲酯的痣,每个脂质的摩尔比计算。例如,要计算的MGDG的摩尔比:

(MGDG)MOL%=Σ[脂肪酸甲酯(MGDG)] /Σ[脂肪酸甲酯(总)] X100%

从野生型和突变导致摩尔比率每个脂质类可以比拟的。例如,tgd4 - 1突变体增加了MGDG和PG的相对含量,但减少DGDG和PE( 5) 18的金额。

图1
图1。极性脂使用拟南芥幼苗的分析流程图。总脂是从4周龄的拟南芥幼苗中提取和薄层色谱法分离。 GLC分析酯交换板薄层色谱分离血脂可刮掉。

图2
图2。血脂薄层板分离。野生型幼苗脂质提取物35毫克(鲜重)是分开的薄层色谱法和硫酸(一),α-萘酚(B)或碘蒸气(三)染色。三重复显示在每个染色法。 DGDG,digalactosyldiacylglycerol; MGDG,monogalactosyldiacylglycerol,PC,磷脂,PE,磷脂,PG,phosphatidylglyce列伊; PI,磷脂酰肌醇; SQDG,sulfoquinovosyldiacylglycerol。

图3
图3。绿灯脂肪酸Methylesters分析(脂肪酸甲酯)来自野生型MGDG。脂肪酸甲酯是一个30米的毛细管柱分离,火焰离子化检测。十五烷酸(15:0)作为内部标准。

图4
图4。COL2野生型(白色列)和tgd4 - 1突变体(黑列)MGDG脂肪酸。脂肪酸的碳双键的数目。三重复的平均值和标准偏差。

图5
图5。极性脂组成的野生型COL2(白色列)和tgd4 - 1突变体(黑列)。 3个重复的平均值和标准偏差错误栏显示。

Discussion

TLC与GLC耦合提供了一个稳健,快速的定量分析植物中的极性脂的工具。脂质成分的微小变化可以识别,因此,这种方法已被用于极性脂代谢途径1,20受损的突变体的大规模筛选。这种方法也被广泛用于监察为底物,利用极性脂的酶活动。2,6,7

除了叶,根和种子或叶绿体和线粒体等亚细胞组分,如其他植物组织的脂质成分也可以以同样的方式确定。

这里使用的溶剂体系(丙酮,甲苯,水)是在植物中的糖脂和磷脂的分离进行了优化。然而,在tgd1,2,3,4突变体和孤立的叶绿体,TGDG运行与PE而tetragalactosyldiacylglycerol与PC机上运行。在这种情况下与氯仿,甲醇,醋酸和水(85:20:10:4,V / V / V / V)的溶剂系统是使用13 。有时二维薄层色谱法,使用两个不同的溶剂体系进行进一步分离糖脂磷脂19。此外,在植物组织中,可直接由绿灯未经薄层色谱法5初步分离,以确定总脂肪酸的名誉反应。除了 ​​表现出的TLC - GLC系统,血脂分析中使用的另一种方法是基于直接电喷雾串联质谱 17 。在此方法中的初始色谱分离提取脂质被省略了。然而,这种方法需要昂贵的设备和经验丰富的人员,这使得常规分析在实验室或突变体筛选有用。

Disclosures

没有利益冲突的声明。

Acknowledgements

这项工作是由美国国家科学基金会克里斯托夫本宁堡的赠款支持。

Materials

Name Company Catalog Number Comments
α-naphthol Sigma-Aldrich N1000
Methanolic HCL 3N Sigma-Aldrich 33050-U Dilute to 1N by methanol
Si250-PA TLC plates JT Baker 7003-04 With pre-absorbent
TLC chamber Sigma-Aldrich Z266000
Screw cap tubes VWR international 53283-800
Scew caps Sun Sri 13-425
PTFE disk Sun Sri 200 608
GLC system Hewlett-Packard HP6890
DB-23 column J&W Scientific 122-2332
GLC vials Sun Sri 500 132
Caps of GLC vials Sun Sri 201 828
Chemstation software Agilent Technologies G2070AA

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References

  1. Ajjawi, I. Large-scale reverse genetics in Arabidopsis: case studies from the Chloroplast 2010 Project. Plant Physiol. 152, 529-529 (2010).
  2. Andersson, M. X., Kjellberg, J. M., Sandelius, A. S. The involvement of cytosolic lipases in converting phosphatidyl choline to substrate for galactolipid synthesis in the chloroplast envelope. Biochim. Biophys. Acta. 1684, 46-46 (2004).
  3. Awai, K. A phosphatidic acid-binding protein of the chloroplast inner envelope membrane involved in lipid trafficking. Proc. Natl. Acad. Sci. U. S. A. 103, e10817-e10817 (2006).
  4. Benning, C. Mechanisms of lipid transport involved in organelle biogenesis in plant cells. Annu. Rev. Cell Dev. Biol. 25, 71-71 (2009).
  5. Browse, J., McCourt, P. J., Somerville, C. R. Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal. Biochem. 152, 141-141 (1986).
  6. Dormann, P., Balbo, I., Benning, C. Arabidopsis galactolipid biosynthesis and lipid trafficking mediated by DGD1. Science. 284, 2181-2181 (1999).
  7. Guskov, A. Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride. 16, 334-334 (2009).
  8. Hartel, H., Dormann, P., Benning, C. DGD1-independent biosynthesis of extraplastidic galactolipids after phosphate deprivation in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 97, 10649-10649 (2000).
  9. James, A. T., MARTIN, A. J. Gas-liquid partition chromatography; the separation and micro-estimation of volatile fatty acids from formic acid to dodecanoic acid. Biochem. J. 50, 679-679 (1952).
  10. Jordan, P. Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature. 411, 909-909 (2001).
  11. Kobayashi, K. Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation. Plant J. 57, 322-322 (2009).
  12. Lu, B. A small ATPase protein of Arabidopsis, TGD3, involved in chloroplast lipid import. J. Biol. Chem. 282, 35945-35945 (2007).
  13. Ohlrogge, J., Browse, J. Lipid biosynthesis. Plant Cell. 7, 957-957 (1995).
  14. Stahl, E. Thin-layer chromatography; methods, influencing factors and an example of its use. Pharmazie. 11, 633-633 (1956).
  15. Stoffel, W., INSULL, W., AHRENS, E. H. Gas-liquid chromatography of highly unsaturated fatty acid methyl esters. Proc. Soc. Exp. Biol. Med. 99, 238-238 (1958).
  16. Welti, R., Wang, X., Williams, T. D. Electrospray ionization tandem mass spectrometry scan modes for plant chloroplast lipids. Anal. Biochem. 314, 149-149 (2003).
  17. Xu, C. Lipid trafficking between the endoplasmic reticulum and the plastid in Arabidopsis requires the extraplastidic TGD4 protein. Plant Cell. 20, 2190-2190 (2008).
  18. Xu, C. Mutation of the TGD1 chloroplast envelope protein affects phosphatidate metabolism in Arabidopsis. Plant Cell. 17, 3094-3094 (2005).
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Comments

9 Comments

  1. Dear All:
    There is something that intrigues me about your protocol, why is formic acid added to the meAdd 300 μL extraction solvent composed of methanol and phosphoric acid added to 1 M potassium chloride (KCl) for the lipid washing part? is this to eliminate contribution of chlorophyll ?

    Reply
    Posted by: Anonymous
    November 4, 2011 - 10:24 AM
  2. It is to kill lipases and prevent breakdown of lipids due to lipase action during extraction.

    Reply
    Posted by: Anonymous
    November 4, 2011 - 10:39 AM
  3. Dear Sir ,
    your presentation is very nice, fruitful, Whether i can use the same method for microalgae lipid extraction. how to understand different lipids or fatty acid from the thin layers ..i mean the names. please give a good solution.

    Reply
    Posted by: Anonymous
    February 7, 2012 - 6:23 AM
  4. Hello,

    Thanks for the question. If there's existing knowledge about the lipid profile of this microalgae, you can simply identify the major lipids on the TLC plate by the relative abundance. You can also use α-naphthol staining to determine the sugar-containing lipids. If it's green algae, the most abundant lipids must be MGDG and DGDG. On the other hand, if no previous knowledge about the lipid profile, scrap off each band after iodine staining and re-extract the lipid with chloroform. Use Mass Spec to determine the identity of each lipid.

    Reply
    Posted by: Anonymous
    February 7, 2012 - 11:52 AM
  5. Dear Sir ,
    thanks for your fruitful presentation.can i use triheptadecanoin as internal standard instead of pentadecanoic acid?How to make sure that the lipid is completely converted into fatty acid methyl ester?

    Reply
    Posted by: wang h.
    March 14, 2013 - 1:43 AM
  6. Hello Dr. Wang,

    To answer your question, you can use triheptadecanoin as internal standard as long as your sample dŒsn't have heptadecanoic acid, which most biological systems don't. But you must make sure to add it before the FAME reaction. Since triheptadecanoin has three C17 FA esterified on a glycerol backbone, during FAME reaction, three molecules of C17 methyester are generated. Therefore during final quantification, you need to divide the molar concentration by 3.
    Regarding the conversion rate of FAMEs, there is no absolute guarantee that all lipids are converted and it is unnecessary because if the internal standards and the samples are treated in parallel, the degree of conversion should be identical.
    I hope this answers your question and let me know if you have any other questions regarding this protocol. Good luck with your experiments!

    Reply
    Posted by: Zhen W.
    March 14, 2013 - 12:17 PM
  7. Dear All,

    Thanks for your nice presentation, it is very helpful. Actually now I am trying to apply this method on green algae, and I got a question regarding the lipid extraction. Our samples are flash frozen in liquid nitrogen and lyophilized, do you think there will be much difference of the result between fresh and lyophilized sample? Do we need to add water to our sample before the extraction? Thanks again!

    Reply
    Posted by: Yan M.
    May 14, 2013 - 2:43 PM
  8. Dear Yan,

    Theoretically, there is no difference between fresh and lyophilized algal sample in terms of lipid extraction. Just be careful during the lyophilization and store in -80C afterward, if not using immediately. If enzymes in cell are not totally deactivated or sample is exposed in room temperature for a long time, unsaturated fatty acids in lipid sample would be easily degraded. There's no need to add water before extraction.

    Good luck with your experiment!

    Bensheng L. and Zhen. W

    Reply
    Posted by: Zhen W.
    May 14, 2013 - 9:22 PM
  9. Thanks for your kind reply. Now we are moving to the step of iodine staining, could you please let me know usually how much iodine is needed to create a saturation of the atmosphere with iodine vapor? We have a similar tank as you showed in the video. Btw, the multiple-plate holder in the tank you showed in the video looks really nice, could you please let me know where did you get this? I searched online but failed to find the proper size. Thanks a lot!

    Reply
    Posted by: Yan M.
    June 28, 2013 - 4:59 PM

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