使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析
Summary
我们提出一个协议，使用液体色谱结合串联质谱法识别和量化主要细胞脂在糖体质酶。所述的酵母细胞内主要脂质类定量评估的方法用途广泛、健壮且敏感。
Abstract
脂质是结构上多样化的两栖分子，不溶于水。脂质是生物膜、能量储存和生产、细胞信号、蛋白质的血管传输、细胞生成和调节细胞死亡的组织和功能的基本贡献者。由于萌芽的酵母糖精，是一种单细胞真核二代，可接受彻底的分子分析，它作为模型有机体的使用有助于揭示脂质代谢和细胞内迁移与真核细胞中复杂的生物过程之间的联系。提供多种分析方法，对酵母细胞内的主要类脂质进行可靠、敏感和准确的定量评估，对于深入了解这些机制至关重要。在这里，我们提出了一个协议，使用液体色谱结合串联质谱（LC-MS/MS）定量分析S.cerevisiae的主要细胞脂质。所述 LC-MS/MS 方法用途广泛且坚固。它能够识别和定量10种脂质中每个物种（包括不同的等体或异质体形式）。该方法很灵敏，允许一些脂质物种在浓度低至0.2pmol/μL的浓度下进行识别和定量。该方法已成功应用于评估全酵母细胞及其纯化细胞体的脂质。该方法采用替代移动相添加剂进行电镀电质谱，可提高部分脂质品种的电理化效率，从而改善其识别和定量。
Introduction
大量证据表明，脂质是生物分子的主要类别之一，在真核细胞的许多重要过程中起着至关重要的作用。这些过程包括组装构成细胞细胞器周围的血浆膜和膜的脂质双层物，在细胞膜之间传输小分子，对细胞外环境变化和细胞内信号转导的反应，能量的产生和储存，限制于不同细胞器的蛋白质的进出口，内膜系统和蛋白质分泌中蛋白质的静脉贩运，以及几种调节细胞死亡模式1 ，2，3，4，5，6，7，8，9，10.
萌芽酵母S.cerevisiae，一个单细胞真核生物，已经成功地揭示了一些机制，脂质在这些重要的细胞过程4，5，6，7，8，9，10，11，12，13，14，15的基本作用，16，17，18，19，20.S. cerevisiae是发现这些机制的宝贵模型有机体，因为它适用于全面的生化、遗传、细胞生物学、化学生物学、系统生物学和微流体解剖分析21、22、23、24、25。脂质代谢和细胞内迁移有助于这些重要细胞过程的相互理解机制的进一步进展，需要敏感的质谱技术，用于细胞脂质的定量表征，了解脂质分子的复杂性，并将定量脂质学整合到系统生物学1、2、3、26的多学科平台中， 27、28、29、30。
目前酵母细胞和其他真核生物细胞的质谱辅助定量脂质学方法不够通用、健壮或敏感。此外，这些目前使用的方法无法区分各种等baric或等质脂质物种。在这里，我们描述了一种多功能、健壮和灵敏的方法，它允许使用液体色谱法与串联质谱（LC-MS/MS）进行定量分析S.cerevisiae的主要细胞脂质。
Protocol
1. 培养酵母的无菌介质的准备 制备含有1%（v/v）酵母提取物和2%（v）百能蛋白的完整YP介质的90 mL。 制备90 mL的合成最小YNB介质，含有0.67%（w/v）酵母氮碱，不含氨基酸，20毫克/L L-西丁，30毫克/L-柳氨酸，30毫克/L-赖氨酸，和20毫克/L尿素。 将 90 mL 的完整 YP 介质平均分成两个 250 mL Erlenmeyer 烧瓶（即每个 45 mL）。 将 90 mL 的合成最小 YNB 介质分成两个 …
Representative Results
在LC-MS/MS的帮助下，我们定量评估酵母细胞内主要细胞脂质的方法是多功能和稳健的。它使我们能够识别和量化10个不同的脂质类在S.cerevisae细胞培养在合成最小YNB培养最初含有2%葡萄糖。这些脂质类包括自由（未酯）脂肪酸（FFA）、CL、植物酰胺（PHC）、植物蛋白酶（PHS）、PC、PE、PG、PI、PS和TAG（补充表1）。使用这种LC-MS/MS方法（补充表1），</stro…
Discussion
以下预防措施对于成功实施此处介绍的协议非常重要：
1. 氯仿和甲醇是有毒的。它们有效地从表面提取各种物质，包括实验室塑料制品和皮肤。因此，请谨慎处理这些有机溶剂，避免在与氯仿和/或甲醇接触的步骤中使用塑料，在这些步骤中使用硼硅酸盐玻璃移液器，并在使用前用氯仿和甲醇对这些移液器进行压毛。
2. 在甲醇/氯仿（17：1）混合物提取脂?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢蒂托连科实验室的现任和前任成员进行讨论。我们感谢质谱生物学应用中心和结构和功能基因组学中心（都位于康科迪亚大学）提供出色的服务。这项研究得到了加拿大自然科学和工程研究理事会（RGPIN 2014-04482）和康科迪亚大学主席基金（CC0113）的资助。K.M.获得康科迪亚大学功绩奖的支持。
Materials
| 15 mL High-speed glass centrifuge tubes with Teflon lined caps | PYREX | 05-550 | |
| 2 mL Glass sample vials with Teflon lined caps | Fisher Scientific | 60180A-SV9-1P | |
| 2-Propanol | Fisher Scientific | A461-500 | |
| Acetonitrile | Fisher Scientific | A9554 | |
| Agilent 1100 series LC system | Agilent Technologies | G1312A | |
| Agilent1100 Wellplate | Agilent Technologies | G1367A | |
| Ammonium acetate | Fisher Scientific | A11450 | |
| Ammonium bicarbonate | Sigma | 9830 | |
| Ammonium formate | Fisher Scientific | A11550 | |
| Ammonium hydroxide | Fisher Scientific | A470-250 | |
| Bactopeptone | Fisher Scientific | BP1420-2 | |
| Cardiolipin | Avanti Polar Lipids | 750332 | |
| Centra CL2 clinical centrifuge | Thermo Scientific | 004260F | |
| Ceramide | Avanti Polar Lipids | 860517 | |
| Chloroform | Fisher Scientific | C297-4 | |
| CSH C18 VanGuard | Waters | 186006944 | Pre-column system |
| Free fatty acid (19:0) | Matreya | 1028 | |
| Glass beads (acid-washed, 425-600 μM) | Sigma-Aldrich | G8772 | |
| Glucose | Fisher Scientific | D16-10 | |
| Hemacytometer | Fisher Scientific | 267110 | |
| L-histidine | Sigma | H8125 | |
| Lipid Search software (V4.1) | Fisher Scientific | V4.1 | LC-MS/MS analysis software |
| L-leucine | Sigma | L8912 | |
| L-lysine | Sigma | L5501 | |
| Methanol | Fisher Scientific | A4564 | |
| Phosphatidylcholine | Avanti Polar Lipids | 850340 | |
| Phosphatidylethanolamine | Avanti Polar Lipids | 850704 | |
| Phosphatidylglycerol | Avanti Polar Lipids | 840446 | |
| Phosphatidylinositol | Avanti Polar Lipids | LM1502 | |
| Phosphatidylserine | Avanti Polar Lipids | 840028 | |
| Reverse-phase column CSH C18 | Waters | 186006102 | |
| Sphingosine | Avanti Polar Lipids | 860669 | |
| Thermo Orbitrap Velos MS | Fisher Scientific | ETD-10600 | |
| Tricylglycerol | Larodan, Malmo | TAG Mixed FA | |
| Ultrasonic sonicator | Fisher Scientific | 15337416 | |
| Uracil | Sigma | U0750 | |
| Vortex | Fisher Scientific | 2215365 | |
| Yeast extract | Fisher Scientific | BP1422-2 | |
| Yeast nitrogen base without amino acids | Fisher Scientific | DF0919-15-3 | |
| Yeast strain BY4742 | Dharmacon | YSC1049 |
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Cite This Article
Mohammad, K., Jiang, H., Hossain, M. I., Titorenko, V. I. Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry. J. Vis. Exp. (157), e60616, doi:10.3791/60616 (2020).