Metabolite and Fatty Acid Analysis of Yeast Cells and Culture Supernatants
酵母细胞和培养上清液中的代谢物和脂肪酸分析
发布: 2014年09月05日第4卷第17期 DOI: 10.21769/BioProtoc.1219 浏览次数: 17775
评审: Claudia CatalanottiAksiniya AsenovaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2014
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Abstract
Metabolite and fatty acid analysis play important roles in evaluating the metabolic state of microorganisms. To examine the growth state and metabolism response of cells to environmental stress or genetic modification, intracellular and extracellular metabolites including fatty acids are usually analyzed to help understand the cellular biochemical changes in microorganisms. In this protocol, gas chromatography-mass spectrometry based analysis was employed to investigate the fatty acids and other metabolites in yeast cells.
Keywords: Metabolite profiling (代谢产物分析)
Fatty acid analysis (脂肪酸分析)
Saccharomyces cerevisiae (酿酒酵母)
Materials and Reagents
- Saccharomyces cerevisiae (S. cerevisiae) cells (EUROSCARF)
- Methanol (Sigma-Aldrich, catalog number: 322415 )
- Ethanol (Sigma-Aldrich, catalog number: 277649 )
- Chloroform (Sigma-Aldrich, catalog number: C2432 )
- Acetic acid (Sigma-Aldrich, catalog number: 320099 )
- Heptadecanoic acid (Sigma-Aldrich, catalog number: H3500 )
- Heptanoic acid (Sigma-Aldrich, catalog number: 75190 )
- Ribitol (Sigma-Aldrich, catalog number: A5502 )
- Fatty acid methyl esters (FAMEs) mix C8-C24 (Sigma-Aldrich, Supelco, catalog number: 18918 )
- 10% BF3-methanol (Fluka, catalog number: 15716 )
- Hexane (Sigma-Aldrich, catalog number: 34859 )
- SPE column (200 mg/3 ml) (Phenomenex, Strata, model: C18-E )
- Adapter (Sigma-Aldrich, Supelco, catalog number: 57020-U )
- Disposable syringe (Sigma-Aldrich, catalog number: Z116866 )
- Sodium chloride (Sigma-Aldrich, catalog number: 746398 )
- Methoxyamine hydrochloride (Sigma-Aldrich, catalog number: 226904 )
- N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) (Fluka, catalog number: 69478 )
- Agilent Technologies Vial Scr Fix Insert Clr PK100 (Agilent, catalog number: 5188-6591 )
- 10 mg/ml heptadecanoic acid (see Recipes)
- 10 mg/ml heptanoic acid (see Recipes)
- 2 mg/ml ribitol (see Recipes)
- Saturated NaCl solution (see Recipes)
Equipment
- Glass beads (acid-washed, 425-600 µm) (Sigma-Aldrich, catalog number: G8772 )
- Fume hood
- SSI 2341-S0S screw cap tubes
- 10 ml PP conical bottom tubes
- HP-5MS capillary column (30 m x 0.250 mm i.d.; film thickness: 0.25 µm) (Agilent)
- Centrifugal Evaporator RC10.09 (Thermo Fisher Scientific, catalog number: 11176780 )
- Thermostatic Cabinet Lovibond ET619-4 (Tintometer GmbH, catalog number: ET2428210 )
- Vortex mixer (CabNet)
- Thermo-shaker
- Eppendorf centriguge 5810R (10,000 rpm) (Eppendorf)
- Thermo scientific Sorvall Legend Micro 21R centrifuge (12,000 rpm-14,000 rpm) (Thermo Fisher Scientific)
- 30 °C Orbital shaker incubator (Yihder Technology, model: LM-570RD )
- NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific)
- FastPrep® -24 instrument (MP Biomedicals, model: 6004-500 )
- Gas chromatography-mass spectrometry (GC-MS) system (Agilent, model: 7890A-5975C ) with an autoinjector G4513A
Software
- Agilent MSD Chemstation data analysis software
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Chen, L. and Chen, W. N. (2014). Metabolite and Fatty Acid Analysis of Yeast Cells and Culture Supernatants. Bio-protocol 4(17): e1219. DOI: 10.21769/BioProtoc.1219.
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分类
微生物学 > 微生物新陈代谢 > 脂质
细胞生物学 > 细胞新陈代谢 > 脂质
生物化学 > 脂质 > 脂质测定
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