Single-cell lipidomics with high structural specificity by mass spectrometry

Zishuai Li,Jing Yang,Wenpeng Zhang,Qiaohong Lin,Minmin Zhang,Aijun Shen,Xiaoxiao Ma,Simin Cheng,Zheng Ouyang,Wenbo Cao,Yu Xia

doi:10.1038/s41467-021-23161-5

Abstract

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. Detailed lipidome characterization for single cells is still far from mature, especially when considering the highly complex structural diversity of lipids and the limited sample amounts available from a single cell. We report the development of a general strategy enabling single-cell lipidomic analysis with high structural specificity. Cell fixation is applied to retain lipids in the cell during batch treatments prior to single-cell analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. Electro-migration combined with droplet-assisted electrospray ionization enables single-cell mass spectrometry analysis with easy operation but high efficiency in sample usage. Four subtypes of human breast cancer cells are correctly classified through quantitative analysis of lipid C=C location or sn-position isomers in ~160 cells. Most importantly, the single-cell deep lipidomics strategy successfully discriminates gefitinib-resistant cells from a population of wild-type human lung cancer cells (HCC827), highlighting its unique capability to promote precision medicine.

Highlights

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis
To extend this method for mammalian cells, isotonic buffer needs to be used to prevent the lysis of the cell membrane, which is incompatible with electrospray ionization
By using precursor ion scan of m/z 184, it was demonstrated that the recovery of PCs after fixation was more than 90% (Fig. 1c, d and Supplementary Fig. 1a–d)

Summary

Introduction

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. MS method coupled with PB reaction were reported to identify C=C locations of lipids in single cell[47], but relative quantitation of isomers has never been done and its unique biological significance was not demonstrated. We develop a protocol with shotgun MS analysis for single-cell lipidomics with high structural specificity including C=C locations or sn-positions (Fig. 1a). The distinct capability of the developed method is that only the relative quantitation of lipid C=C location isomers can discriminate gefitinib-resistant single cells in wild-type non-smallcell lung cancer (NSCLC) cell population, demonstrating its unique significance for precision medicine

Methods

Results

Conclusion