High-throughput single-cell mass spectrometry enables metabolic network analysis by resolving phospholipid C=C isomers

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 (C=C) location isomer resolution analysis, aided by C=C activation in unsaturated lipids by the Patern & ograve;-B & uuml;chi (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 C=C from 5 lipid precursors was determined. While cell-to-cell variations in MS1-based lipid profiling can be large, an advantage of quantifying lipid C=C location isomers is the significantly improved quantitation accuracy. For example, the relative standard deviations (RSDs) of the relative amounts of PC 34:1 C=C 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.