Exploration of Single Cell Lipidomics with a Novel Multi-reflecting Q-Tof Platform

Importance of the Topic

The ability to profile lipids at the single-cell level is revolutionizing our understanding of cellular heterogeneity in health and disease. Advances in mass spectrometry sensitivity, resolution and speed now enable detailed lipidomic analyses on individual cells, providing insights into metabolic diversity, signaling pathways and responses to treatments that would be masked in bulk measurements.

Aims and Overview of the Study

This work presents a novel single-cell lipidomics workflow using a multi-reflecting quadrupole time-of-flight platform. The study evaluates chromatographic and mass spectrometric parameters to achieve robust separation and high sensitivity. Three human cell lines (THP-1, C1R and Jurkat) serve as biological models to demonstrate method performance across dilute bulk extracts and fluorescence-activated cell sorting (FACS)-isolated cells down to a single cell.

Methodology and Instrumentation

The workflow combines an ACQUITY Premier liquid chromatography system with a Xevo MRT mass spectrometer. Key optimization steps included:

• Column chemistries compared: C18, C30 and C8 stationary phases
• Gradient durations from 3.7 to 29 minutes to balance throughput and resolution
• Flow rates between 0.1 and 0.4 mL/min
• Scan speeds of 1, 5, 10 and 20 Hz for data acquisition

EquiSPLASH® lipid standards were used in a dilution series (0.5–1000 ng/mL) to benchmark LOD (5.5 ng/mL) and LOQ (16.4 ng/mL) for 18:1(d7) LPC, yielding a calibration with r2 of 0.9999. Cell samples were prepared both as bulk extracts at concentrations from 10,000 to 1 cell/µL and as sorted single-cell, 10-cell and 100-cell groups in 96-well plates spiked with internal standards.

Key Results and Discussion

The optimized method achieved sub-ppm mass accuracy (<1 ppm) and detected a broad range of lipid classes across five orders of magnitude in dynamic range. Identified lipids included phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, ceramides, triacylglycerols and cholesteryl esters. Principal component analyses demonstrated clear discrimination between cell types and cell counts, confirming the workflow’s reproducibility and sensitivity for single-cell lipidomics. Matched MS/MS fragmentation further validated lipid assignments with high confidence scores (>89%).

Benefits and Practical Applications

This approach offers:

• High sensitivity to profile lipids from individual cells
• Fast and robust LC-MS workflow suitable for high-throughput studies
• Accurate quantitation over a wide dynamic range
• Compatibility with FACS for targeted cell selection

Applications span immunology, cancer research, drug screening and quality control in biomanufacturing, where understanding cell-to-cell variability is critical.

Future Trends and Applications

Ongoing developments may include integration with ion mobility to resolve isomeric lipids, automation of single-cell sample handling, and expansion to multi-omics by combining lipidomics with proteomics or transcriptomics. Enhanced data analysis platforms featuring machine learning will further streamline feature annotation and biological interpretation.

Conclusion

The presented single-cell lipidomics workflow on a multi-reflecting Q-Tof platform demonstrates exceptional resolution, sensitivity and dynamic range. By coupling optimized LC conditions with a high-performance mass spectrometer and robust data analysis, lipid profiling at the individual cell level becomes feasible for diverse research and industrial applications.

References

1. Pang Z., Lu Y., Zhou G., Hui F., Xu L., Viau C., Spigelman A., MacDonald P., Wishart D., Li S., Xia J. MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Research (2024).