In Situ Microprobe Single-Cell Capillary Electrophoresis Mass Spectrometry: Metabolic Reorganization in Single Differentiating Cells in the Live Vertebrate (Xenopus laevis) Embryo.
In Situ Microprobe Single-Cell Capillary Electrophoresis Mass Spectrometry: Metabolic Reorganization in Single Differentiating Cells in the Live Vertebrate (Xenopus laevis) Embryo.
复制标题
DOI：
10.1021/acs.analchem.7b00880
复制
发表时间：
2017-07-05
影响因子：
7.4
通讯作者：
Nemes P
中科院分区：
文献类型：
--
作者：
Onjiko RM;Portero EP;Moody SA;Nemes P
单细胞代谢的知识将提供一个强大的细胞活性变化的细胞分化为脊椎动物胚胎的所有组织。然而，单细胞质谱技术尚未与胚胎早期发育期间复杂的三维变化和迅速减小的细胞尺寸兼容。在这里，我们通过整合毛细管微量取样，微量代谢物提取和毛细管电泳电喷雾电离质谱（CE-ESI-MS），使直接代谢分析活青蛙胚胎（非洲爪蟾）中识别的细胞，弥合了这一技术差距。<0.02%的单细胞含量的微探针CE-ESI-MS使我们能够在8至32细胞胚胎的单个背侧和腹侧细胞中检测到大约230种不同的分子特征（正离子模式），包括70种已知的代谢物。相对定量，然后进行多变量和统计分析的数据发现，微量采样提高检测灵敏度相比，全细胞解剖，最大限度地减少化学干扰和离子抑制效应的培养基。此外，更高的谷胱甘肽/氧化型谷胱甘肽的比例表明，微探针细胞表现出显着较低的氧化应激比从胚胎解剖。快速（5秒/细胞）和可扩展的微量取样，对8细胞胚胎中的细胞损伤最小，能够对同一细胞进行一式两份和一式三份的代谢分析，该细胞令人惊讶地继续分裂到16细胞阶段。最后，我们使用微探针单细胞CE-ESI-MS来揭示先前未知的单细胞代谢组的重组，因为来自8细胞胚胎的背祖细胞通过分裂到32细胞胚胎形成了神经组织命运克隆，第一次窥视到脊椎动物胚胎早期发育过程中代谢单细胞异质性的形成。
Knowledge of single-cell metabolism would provide a powerful look into cell activity changes as cells differentiate to all the tissues of the vertebrate embryo. However, single-cell mass spectrometry technologies have not yet been made compatible with complex three-dimensional changes and rapidly decreasing cell sizes during early development of the embryo. Here, we bridge this technological gap by integrating capillary microsampling, microscale metabolite extraction, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to enable direct metabolic analysis of identified cells in the live frog embryo (Xenopus laevis). Microprobe CE-ESI-MS of <0.02% of the single-cell content allowed us to detect ∼230 different molecular features (positive ion mode), including 70 known metabolites, in single dorsal and ventral cells in 8-to-32-cell embryos. Relative quantification followed by multivariate and statistical analysis of the data found that microsampling enhanced detection sensitivity compared to whole-cell dissection by minimizing chemical interferences and ion suppression effects from the culture media. In addition, higher glutathione/oxidized glutathione ratios suggested that microprobed cells exhibited significantly lower oxidative stress than those dissected from the embryo. Fast (5 s/cell) and scalable microsampling with minimal damage to cells in the 8-cell embryo enabled duplicate and triplicate metabolic analysis of the same cell, which surprisingly continued to divide to the 16-cell stage. Last, we used microprobe single-cell CE-ESI-MS to uncover previously unknown reorganization of the single-cell metabolome as the dorsal progenitor cell from the 8-cell embryo formed the neural tissue fated clone through divisions to the 32-cell embryo, peering, for the first time, into the formation of metabolic single-cell heterogeneity during early development of a vertebrate embryo.
登录
查看更多内容
影响因子：
--
作者：
Ichu TA;Han J;Borchers CH;Lesperance M;Helbing CC
通讯作者：
Helbing CC
影响因子：
2.5
作者：
Grant, Paaqua A.;Yan, Bo;Moody, Sally A.
通讯作者：
Moody, Sally A.
影响因子：
10
作者：
DEUCHAR, EM
通讯作者：
DEUCHAR, EM
DOI：
10.1007/s13361-014-0978-9
发表时间：
2014-11-01
影响因子：
3.2
作者：
Lanni, Eric J.;Dunham, Sage J. B.;Sweedler, Jonathan V.
通讯作者：
Sweedler, Jonathan V.
影响因子：
7
作者：
Lombard-Banek, Camille;Reddy, Sushma;Nemes, Peter
通讯作者：
Nemes, Peter