Selective Capture-Based Single-Cell Mass Spectrometry for Enhancing Sphingolipid Profiling of Neurons with...

Currently, single-cell lipidomic mass spectrometry (MS) techniques are mostly limited to detection of high-abundance phosphatidylcholines (PCs). Herein, for enhancing the coverage to low-abundance sphingolipids in single-cell analysis, in-tube solid-phase microextraction (SPME) was combined with a single-probe MS system for selective enrichment of sphingolipids during singe-cell sampling. From the results, a lab-made single probe with a 30 μm tip size proved to be able to resolve the axon from the cell body of neuron HT22 in ambient conditions. TiO₂ was immobilized onto the inner wall of the transfer capillary of the single-probe device for online selective capture of sphingolipids in ammonia-acetonitrile and rapid desorption in formic acid-methanol. The results showed that the breakthrough volume of the capillary with sample loading flow rate at 500 nL/min was >14 μL. Standard experiments showed that the signals of cerebroside (CB), ceramide (Cer), and sphingomyelin (SM) were largely enhanced after selective capture in the coated capillary, while PCs were totally removed. The reusability (>10 times) and stability of the lab-made TiO₂-coated capillary was verified. By introducing the coated capillary into the single-probe MS system, the new system proved to have low detection limits of SM, Cer, and CB (0.007-0.027 μg/mm²) and acceptable linearity (r > 0.98) and repeatability (RSD < 30%). Lipid coverage of the new method to SMs and CBs proved to be largely improved (SM, 21 vs 2; CB, 10 vs 0) with the new method in comparison to conventional single-probe MS without selective capture by ambient analysis of a single spot of rat cerebellum. Finally, the new system was used to perform single-neuron analysis of sphingolipids in the control and lipopolysaccharide (LPS)-treated HT22 with differentiation of the cell body from the axonal synapse. Results showed that 5 sphingolipids had significantly higher concentrations in the synapse than in the cell body, while 3 oxidized sphingolipids had significantly higher levels in the cell body than in the synapse. After LPS treatment, most of the sphingolipids largely decreased and became more accumulated in the synapse, providing new information on LPS-induced neuroinflammation.