Extraction and Profiling of Plant Polar Glycerol Lipids
植物极性甘油脂质的提取和分析
发布: 2016年06月20日第6卷第12期 DOI: 10.21769/BioProtoc.1849 浏览次数: 12044
评审: Marisa RosaAgnieszka ZienkiewiczAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
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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
- PYREXTM screw cap culture tubes with PTFE lined phenolic caps (Thermo Fisher Scientific, catalog number: 14-933D )
- Disposable glass pipettes (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: 13-678-20C )
- Autosampler vials (Sigma-Aldrich, catalog number: 29133-U ) with pre-cut cap (Sigma-Aldrich, catalog number: 29494-U )
- TLC Silica gel 60 plate (Merck)
- 1 ml syringe
- Injection loop
- 4-6 weeks old Arabidopsis rosette leaves
Note: Other plant materials can be used, additional precautions may be needed (see Notes). - Isopropanol (Thermo Fisher Scientific, catalog number: A416 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
- Chloroform (Thermo Fisher Scientific, catalog number: C607 )
- Methanol (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: A456 )
- Acetic acid
- Iodine
- Primuline
- Acetone
- Nitrogen gas
- Butylated hydroxytoluene (BHT) (Sigma-Aldrich, catalog number: B1378 )
- 7.5 M ammonium acetate solution (NH4Ac) (Sigma-Aldrich, catalog number: A2706 )
- Phospholipid (PL) standard [kindly provided by Dr. Ruth Welti, (Kansas State University)]
Note: An example PL standard list is shown in Table 1. - Monogalactosyldiacylglycerol (MGDG), hydrogenated (Matreya, catalog number: 1058 )
- 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). - Isopropanol solution with BHT (see Recipes)
- Lipid extraction solution (see Recipes)
- KCl solution (see Recipes)
- Solution B (see Recipes)
- Galactolipid (GL) standard (see Recipes)
- Phospholipid (PL) standard (see Recipes)
Equipment
- Water bath (Thermo Fisher Scientific, model: Isotemp2340 )
- Water bath shaker (Labnet International, model: SWB5050 )
- Nitrogen evaporator N-EVAP (Organomation, model: 111 )
- Fume hood
- Ultraviolet light (360 nm)
- Oven (Thermo Fisher Scientific, Fisher ScientificTM, model: 13-247-650G )
- 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
- Analyst 1.5.1 (AB SCIEX)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Liu, Y. and Wang, X. (2016). Extraction and Profiling of Plant Polar Glycerol Lipids. Bio-protocol 6(12): e1849. DOI: 10.21769/BioProtoc.1849.
Download Citation in RIS Format
分类
生物化学 > 脂质 > 脂质分离
生物化学 > 脂质 > 脂质测定
植物科学 > 植物生物化学 > 脂质
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