Highly Sensitive Targeted Method for Single Cell Lipidomics on Stellar MS – A Hybrid Nominal Mass Instrument

Importance of the Topic

Lipid molecules are fundamental components of cellular structure and signaling. Their diverse roles in metabolism and disease highlight the need for detailed profiling at the single-cell level. Traditional bulk analyses average signals across populations, masking cellular heterogeneity and rare subpopulations. Single-cell lipidomics offers a path to uncover unique lipid signatures, enabling insights into development, disease progression, and therapeutic responses.

Objectives and Study Overview

This study aimed to develop a highly sensitive, targeted workflow for single-cell lipidomics using the Thermo Scientific Stellar mass spectrometer. Key goals included enhancing specificity, improving detection limits, and increasing throughput compared to existing untargeted methods. Researchers extracted lipids from both bulk and individual RK13 and Vero cells, established a targeted lipid list, and evaluated performance across instruments.

Methodology

The workflow comprised:

• Sample preparation: Lipid extraction from bulk cell pellets (100 cells) and isolated single cells of two cell lines.
• Separation: Nano‐UHPLC with a C18 column for high‐resolution chromatographic separation.
• Data acquisition: Untargeted data‐dependent acquisition on an Orbitrap Ascend Tribrid MS; targeted parallel reaction monitoring on the Stellar MS using an 864‐lipid inclusion list derived from bulk data.
• Data processing: Compound Discoverer 3.4, LipidSearch, and Skyline for annotation, quantitation, and isomer discrimination through multi‐stage fragmentation and iterative acquisition workflows.

Instrumentation

• Thermo Scientific Vanquish Neo UHPLC system with EASY‐Spray PepMap Neo capillary column (C18, 75 µm x 150 mm, 2 µm).
• Thermo Scientific Orbitrap Ascend Tribrid mass spectrometer for untargeted DDA.
• Thermo Scientific Stellar MS nominal‐mass instrument for targeted nanoLC‐MS analysis.
• AcquireX intelligent data acquisition workflow for bulk sample annotation enhancement.

Main Results and Discussion

A targeted lipid list of 864 entries was generated from bulk extracts, with 613 isomeric precursors optimized for Stellar MS. Nano‐flow separation enhanced sensitivity, enabling detection down to tens of picograms per cell. Single‐cell analyses identified 258 lipids across RK13 and Vero cells, revealing distinct lipid profiles and clear separation by principal component analysis. Multi‐stage MSn fragmentation improved confidence in isomer assignments and reduced false positives.

Benefits and Practical Applications

The targeted approach on Stellar MS offers:

• High sensitivity and specificity for low‐abundance lipids in single cells.
• Cost‐effective instrumentation compared to high‐resolution platforms.
• Quantitative capabilities for biomarker discovery and cell‐type classification.
• Streamlined data processing with established software packages.

Future Trends and Opportunities

Anticipated developments include:

• Integration with single‐cell transcriptomics and proteomics for multi‐omics mapping.
• AI‐driven annotation pipelines to accelerate lipid identification.
• Expanded targeted panels for disease‐specific lipid biomarkers.
• Improved throughput via microfluidic sample handling and multiplexed acquisition.

Conclusion

This work demonstrates a robust, targeted lipidomics workflow for single‐cell analysis using the Stellar MS platform. The method achieves deep lipid coverage, high sensitivity, and reliable quantitation, paving the way for broader adoption in research and clinical laboratories.

References

• Deshpande RR, Liu Y, Amer B, Gaul DA, Fernandez F, Bird S. Highly Sensitive Targeted Method for Single Cell Lipidomics on Stellar MS. Thermo Fisher Scientific; Georgia Institute of Technology; 2025.