Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis-Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration
Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis-Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration
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DOI：
10.1021/acs.analchem.9b01578
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发表时间：
2019-08-20
影响因子：
7.4
通讯作者：
Tanaka, Yo
中科院分区：
文献类型：
--
作者：
Kawai, Takayuki;Ota, Nobutoshi;Tanaka, Yo
单细胞代谢组分析对于研究神经网络和肿瘤微环境等微观生命现象是必不可少的。毛细管电泳质谱(CE-MS)是最灵敏的技术之一，但其灵敏度仍不足以满足一般人体细胞(如HeLa)的单细胞分析。为了解决这些问题，我们首先开发了一种高效的电离发射器，名为“NanCESI”发射器，具有薄壁(类似于10微米)和锥形(5-10微米)的末端。薄导电壁实现了无护套电离，最大限度地降低了电离样品的流速，锥形端通过电喷雾电离机制有效地电离了分析物，与传统的无护套发射器相比，灵敏度提高了3.5倍。利用纳米CESI发射器成功地实现了对20种氨基酸的50次重复分析。50种分析方法对迁移时间、峰高和峰面积的相对标准偏差分别为1.5%、4.4%和6.8%，检出限为170 pm(850zmoL)。其次，将大容量等速电泳法和堆积法(LDIS)的样品富集法应用于一种新设计的纳米CESI-MS分析方法。该方法实现了高达380倍的灵敏度和450 FM的LOD。与普通的无鞘CE-MS相比，纳米CESI和LDIS的联用使总的灵敏度提高了800倍。最后，对单个HeLa细胞进行了代谢组学分析，其中20个氨基酸被成功地用三重四极杆MS定量，40个代谢物被用四极杆飞行时间质谱仪鉴定，为下一代的微型生物分析提供了一个有前景的分析平台。
Single cell metabolome analysis is essential for studying microscale life phenomena such as neuronal networks and tumor microenvironments. Capillary electrophoresis-mass spectrometry (CE-MS) is one of the most sensitive technologies; however, its sensitivity is still not enough for single cell analysis on general human cells such as HeLa. To address these issues, we first developed an efficient ionization emitter, named as a "nanoCESI" emitter, that had a thin-walled (similar to 10 mu m) and tapered (5-10 mu m) end. The thin conductive wall enabled sheathless ionization and minimized the flow rate of ionizing sample, and the tapered end efficiently ionized analytes via an electrospray ionization mechanism, providing up to 3.5-fold increase in sensitivity compared with a conventional sheathless emitter. Fifty repetitive analyses on 20 amino acids were successfully achieved with a nanoCESI emitter. Relative standard deviations of 50 analyses were 1.5%, 4.4%, and 6.8% for migration time, peak height, and peak area, respectively, where a limit of detection (LOD) of 170 pM (850 zmol) was achieved. Second, a sample enrichment method, large-volume dual preconcentration by isotachophoresis and stacking (LDIS), was applied to a newly designed protocol of nanoCESI-MS. This approach achieved up to 380-fold enhanced sensitivity and LOD of 450 fM. Compared with normal sheathless CE-MS, coupling of nanoCESI and LDIS provided up to 800-fold increase of sensitivity in total. Finally, metabolome analyses of single HeLa cells were performed, where 20 amino acids were successfully quantified with triple-quadrupole MS and 40 metabolites were identified with quadrupole-time-of-flight MS, as a promising analytical platform for microscale bioanalysis for the next generation.