Single-cell mass spectrometry (MS) is an essential technology for sensitive and multiplexed analysis of metabolites and lipids for cell phenotyping and pathway studies. However, the structural elucidation of lipids from single cells remains a challenge, especially in the high-throughput scenario. Technically, there is a contradiction between the inadequate sample amount (i.e. a single cell, 0.5–20 pL) for replicate or multiple analysis, on the one hand, and the high metabolite coverage and multidimensional structure analysis that needs to be performed for each single cell, on the other hand. Here, we have developed a high-throughput single-cell MS platform that can perform both lipid profiling and lipid carbon–carbon double bond (CC) location isomer resolution analysis, aided by CC activation in unsaturated lipids by the Paternò–Büchi (PB) reaction and tandem MS, termed single-cell structural lipidomics analysis. The method can achieve a single-cell analysis throughput of 51 cells per minute. A total of 145 lipids were structurally characterized at the subclass level, of which the relative abundance of 17 isomeric lipids differing in the location of CC from 5 lipid precursors was determined. While cell-to-cell variations in MS¹-based lipid profiling can be large, an advantage of quantifying lipid CC location isomers is the significantly improved quantitation accuracy. For example, the relative standard deviations (RSDs) of the relative amounts of PC 34:1 CC position isomers in MDA-MB-468 cells are half smaller than those measured for PC 34:1 as a whole by MS¹ abundance profiling. Taken together, the developed method can be effectively used for in-depth structural lipid metabolism network analysis by high-throughput analysis of 142 MDA-MB-468 human breast cancer cells.

中文翻译：

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高通量单细胞质谱通过解析磷脂 CC 异构体​​实现代谢网络分析

单细胞质谱 (MS) 是对代谢物和脂质进行灵敏、多重分析的重要技术，可用于细胞表型分析和通路研究。然而，单细胞脂质的结构解析仍然是一个挑战，特别是在高通量情况下。从技术上讲，一方面用于重复或多重分析的样品量不足（即单个细胞，0.5-20 pL），另一方面需要对每个单个细胞进行高代谢物覆盖和多维结构分析。另一方面，细胞。在这里，我们开发了一个高通量单细胞 MS 平台，可以在 Paternò-Büchi 不饱和脂质中的 C C 激活的帮助下进行脂质分析和脂质碳碳双键 (CC ) 位置异构体分辨率分析。PB) 反应和串联 MS，称为单细胞结构脂质组学分析。该方法可实现每分钟51个细胞的单细胞分析通量。总共 145 种脂质在亚类水平上进行了结构表征，其中 17 种异构脂质的相对丰度被确定，这些异构体脂质的 C C 位置与 5 种脂质前体不同。虽然基于 MS ¹的脂质分析中的细胞间差异可能很大，但定量脂质 C C 位置异构体的一个优点是显着提高了定量精度。例如，MDA-MB-468 细胞中 PC 34:1 C C 位异构体相对量的相对标准偏差 (RSD)比通过 MS ¹丰度分析对 PC 34:1 整体测量的值小一半。综上所述，所开发的方法可以通过对 142 个 MDA-MB-468 人乳腺癌细胞进行高通量分析，有效地用于深入的结构脂质代谢网络分析。