Selective Capture-Based Single-Cell Mass Spectrometry for Enhancing Sphingolipid Profiling of Neurons with Differentiation of Cell Body from Synapse

Abstract

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 solidphase 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. TiO 2 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 TiO 2 -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/mm 2 ) 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.

Single-Cell Lipidomics Using Analytical Flow LC-MS Characterizes the Response to Chemotherapy in Cultured Pancreatic Cancer Cells

“…Capillary sampling under microscope observation followed by nanoelectrospray ionization (NSI) is a technique that has been employed for the detection of metabolites and lipids in single living cells. − This approach is advantageous because it can isolate live single cells in their native state, and spatial information is retained through using microscopy. , However, NSI suffers from challenges with precision and lacks automation, both in data acquisition and analysis.…”

Pulled Flowprobe for Ambient Liquid Extraction-Based High Spatial Resolution Mass Spectrometry Imaging with Enhanced Sensitivity and Stability

“…17−19 Intube solid-phase microextraction was introduced into singleprobe MS to achieve online selective capture during single-cell analysis in our previous study. 20 Also, ambient liquid extraction coupled with ESI-MS has benefits on proteoform imaging due to higher charge states of protein ions which is advantageous for on-tissue top-down proteomics. 21 Thus, ambient liquid extraction techniques have been widely used in spatial metabolomics and lipidomics and spatial proteomics.…”

“…For example, online photochemical reaction for lipid isomer identification through selective derivatization of double bonds was introduced into nano-DESI . Standards were easily doped into the extraction solvents for different standard calibration methods. − In-tube solid-phase microextraction was introduced into single-probe MS to achieve online selective capture during single-cell analysis in our previous study . Also, ambient liquid extraction coupled with ESI-MS has benefits on proteoform imaging due to higher charge states of protein ions which is advantageous for on-tissue top-down proteomics .…”

Inner-Wall Coated Nanopipette Microextraction for Quantitative Analysis of Per- and Polyfluoroalkyl Substances in Single Cells Using Mass Spectrometry

“…26 A TiO 2 -coated in-tube SPME combined with single-probe MS system was developed to selectively enrich the sphingolipids and enhance the coverage of low-abundance sphingolipids in single-cell analysis. 27 Here we developed an inner-wall coated nanopipette microextraction coupled with InESI-MS method for sensitive and accurate analysis of trace PFASs and investigation of their accumulative behaviors and heterogeneities in single cells. A specially designed inner-wall-coated nanopipette, with the modification of a coating containing octadecyl, phenyl, primary amine, and quaternary amine groups on the inner wall, was designed and prepared for sampling of the cytoplasm from a single cell.…”

“…For instance, a microscale SPME probe with a tip-end diameter of ∼5 μm was developed to enrich lipids from single HepG2 cells for single-cell MS analysis . A TiO 2 -coated in-tube SPME combined with single-probe MS system was developed to selectively enrich the sphingolipids and enhance the coverage of low-abundance sphingolipids in single-cell analysis …”