Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo.
Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo.
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DOI：
10.1039/c6an00200e
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发表时间：
2016-06-21
期刊：
影响因子：
--
通讯作者：
Nemes P
中科院分区：
文献类型：
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作者：
Onjiko RM;Morris SE;Moody SA;Nemes P
单细胞代谢质谱能够发现（非靶向）分析单个细胞中的小分子。使用单细胞毛细管电泳高分辨率质谱（CE-HRMS），我们最近发现位于沿着动物-植物和背腹轴的16细胞青蛙（非洲爪蟾）胚胎的胚胎细胞之间的小分子差异，提出了代谢细胞异质性是否也存在沿着左右体轴的问题。为了解决这个问题，我们在这里提出了单细胞CE-HRMS，用于以更高的分析灵敏度鉴定和定量代谢物，然后使用该方法来比较左细胞和右细胞之间的代谢物产生。我们的策略利用具有互补物理化学性质的多种溶剂从单细胞中提取小分子并改善电泳分离，增加代谢物离子信号用于定量和串联HRMS。结果，我们能够在从8细胞胚胎中分离的D1细胞中鉴定出55种不同的小分子。为了定量左细胞和右细胞之间的代谢产物产生，我们在技术重复测量中分析了n = 24个不同的D1细胞。基于80种最重复定量的化合物的统计学和多变量分析揭示了10种不同的代谢物，其在左侧或右侧细胞中显著差异地积累（p < 0.05并且倍数变化≥ 1.5）。这些代谢产物在右侧的精氨酸-脯氨酸代谢途径中富集，但在左侧D1细胞中不富集。除了为单细胞HRMS提供分析优势外，这项工作还为在早期发育胚胎中建立正常身体不对称性提供了新的代谢数据。
Single-cell metabolic mass spectrometry enables the discovery (untargeted) analysis of small molecules in individual cells. Using single-cell capillary electrophoresis high-resolution mass spectrometry (CE-HRMS), we recently uncovered small-molecule differences between embryonic cells located along the animal–vegetal and dorsal–ventral axes of the 16-cell frog (Xenopus laevis) embryo, raising the question whether metabolic cell heterogeneity also exists along the left–right body axis. To address this question, we here advance single-cell CE-HRMS for identifying and quantifying metabolites in higher analytical sensitivity, and then use the methodology to compare metabolite production between left and right cells. Our strategy utilizes multiple solvents with complementary physicochemical properties to extract small molecules from single cells and improve electrophoretic separation, increasing metabolite ion signals for quantification and tandem HRMS. As a result, we were able to identify 55 different small molecules in D1 cells that were isolated from 8-cell embryos. To quantify metabolite production between left and right cells, we analyzed n = 24 different D1 cells in technical duplicate–triplicate measurements. Statistical and multivariate analysis based on 80 of the most repeatedly quantified compounds revealed 10 distinct metabolites that were significantly differentially accumulated in the left or right cells (p < 0.05 and fold change ≥ 1.5). These metabolites were enriched in the arginine–proline metabolic pathway in the right, but not the left D1 cells. Besides providing analytical benefits for single-cell HRMS, this work provides new metabolic data on the establishment of normal body asymmetry in the early developing embryo.