An Analytical System for Single-Cell Metabolomics of Typical Mammalian Cells Based on Highly Sensitive Nano-Liquid Chromatography Tandem Mass Spectrometry.

Q3 Physics and Astronomy
Mass spectrometry Pub Date : 2020-01-01 Epub Date: 2020-03-17 DOI:10.5702/massspectrometry.A0080
Kohta Nakatani, Yoshihiro Izumi, Kosuke Hata, Takeshi Bamba
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引用次数: 15

Abstract

The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10-20 μm are still in the process of development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-μm particle-size pentafluorophenylpropyl Discovery HSF5 of dimensions 100 μm i.d.×180 mm) was prepared using a slurry technique. The optimized nano-LC-MS/MS method showed 3-132-fold (average value, 26-fold) greater sensitivity than semimicro-LC-MS/MS, and the detection limits for several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC-MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabolites) from single HeLa cells (n=22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity.

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基于高灵敏度纳米液相色谱串联质谱的典型哺乳动物细胞单细胞代谢组学分析系统。
新一代测序技术的快速发展使得单细胞基因组学和转录组学分析成为可能,这揭示了生物系统异质性的重要性。然而,从典型直径为10-20 μm的单个哺乳动物细胞中准确鉴定和定量综合代谢物的分析方法仍在发展中。本研究的目的是建立一种基于高灵敏度纳米液相色谱串联质谱(nano-LC-MS/MS)的单细胞代谢组学分析系统,并进行多反应监测。采用浆液法制备了粒径为3 μm、尺寸为100 μm i.d.×180 mm的五氟苯基丙基发现HSF5填充纳米色谱柱。优化后的纳米lc -MS/MS方法的灵敏度比半微lc -MS/MS高3-132倍(平均值26倍),对氨基酸和核酸相关代谢物等几种亲水性代谢物的检出限在亚fmol范围内。通过单细胞活样和纳米lc -MS/MS相结合,我们成功地从单个HeLa细胞(n=22)中检测到18种相对丰富的亲水性代谢物(16种氨基酸和2种核酸相关代谢物)。根据单细胞代谢谱,22个HeLa细胞被分为三个不同的亚类,这表明培养的HeLa细胞群体的代谢功能存在差异。我们的单细胞代谢组学分析系统为深入研究细胞代谢和异质性提供了一个潜在的有用工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Mass spectrometry
Mass spectrometry Physics and Astronomy-Instrumentation
CiteScore
1.90
自引率
0.00%
发文量
3
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