Automated Dual Metabolite+Lipid Cell Sample Prep as a Component of an LC/HRMS-based Workflow to Elucidate the Molecular Response to Drug Treatment

Importance of the Topic

The ability to extract both polar metabolites and lipids from a single population of mammalian cells under ambient conditions is critical for comprehensive molecular profiling. Such dual extraction workflows enhance reproducibility, reduce sample consumption, and provide integrated metabolic and lipidomic views of cellular responses to perturbations, particularly in oncology and drug discovery contexts.

Study Objectives and Overview

This work introduces an automated, room-temperature cell lysis and quench protocol coupled with sequential polar metabolite and lipid fractionation. Using K562 acute myeloid leukemia cells treated with methotrexate (MTX) or saline control, the study aims to demonstrate:

• High recovery and low variability for both metabolite and lipid classes.
• Compatibility with liquid-handling automation and high-resolution LC/Q-TOF analysis.
• Integrated untargeted metabolomics and lipidomics data workflows to uncover MTX-induced molecular changes.

Methodology and Instrumentation

Sample Preparation and Extraction:

• Cultured K562 cells (1×10^6 per well) were treated with 1 µM MTX or vehicle for 24 h, then lysed and metabolism quenched using 50 % trifluoroethanol at ambient temperature.
• Dual extraction employed Captiva EMR-lipid plate for protein removal, polar metabolite elution, plate wash, and subsequent lipid elution by solvent switch.
• 13C-labeled yeast extract standards and 2H-labeled lipid mixtures spiked into parallel samples to monitor recovery and reproducibility.
• Automated processing executed on an Agilent Bravo Liquid Handling Platform.

LC/HRMS and Data Processing:

• Polar metabolites separated by reversed-phase ion-pairing LC; lipids by reversed-phase LC in both positive and negative electrospray modes.
• Detection on Agilent 6546 LC/Q-TOF instrument.
• Data analysis via Agilent MassHunter Quantitative Analysis, Profinder for batch feature extraction, Lipid Annotator for MS/MS annotation, and Mass Profiler Professional (MPP) for statistical and clustering analyses.

Main Results and Discussion

Recovery and Precision:
Overall recovery averaged 95 % for polar metabolites (87 % within 90–110 %) and 85 % for lipids across classes, with median %RSD < 6.5 % for both assays, demonstrating high reproducibility.

Metabolomics Findings:

• Untargeted profiling identified ~4,700 features; 458 showed significant MTX-induced changes (fold-change ≥1.5, p < 0.05).
• Pyrimidine and purine pathway intermediates accumulated, reflecting MTX inhibition of folate-dependent enzymes.

Lipidomics Findings:

• Iterative MS/MS annotation yielded a comprehensive lipid library.
• MTX treatment correlated with increased ceramides and acylcarnitines and decreased short-chain phosphatidylinositols, suggesting shifts in sphingolipid metabolism.

Integrated Multi-Omics Correlation:
Correlation analysis in MPP revealed strong positive correlations (r > 0.9) between metabolites such as L-serine and ceramide species, indicating coordinated alterations in amino acid and sphingolipid biosynthesis under MTX stress.

Practical Applications and Benefits

• Streamlined, single-sample workflow reduces processing time and sample requirements.
• Automated protocol enhances throughput and consistency.
• Ambient-temperature lysis and quench preserve labile metabolites.
• Integrated data analysis enables direct cross-comparison of metabolomic and lipidomic changes.

Future Trends and Potential Applications

Advancements in automation and microfluidics may further miniaturize sample prep and increase throughput for clinical and pharmaceutical screening. Integration with machine-learning algorithms and expanded spectral libraries will improve annotation confidence and pathway mapping. The approach could be extended to primary patient samples and multiplexed drug screening for personalized medicine applications.

Conclusion

This study demonstrates a robust, automated dual extraction workflow for combined metabolomic and lipidomic analysis of mammalian cells. The protocol delivers high recovery, low variability, and seamless integration with LC/HRMS and bioinformatics platforms, providing deep insights into cellular responses to chemotherapeutic treatment.

Reference

1. Spivia W, et al. Automated Metabolite Extraction for Plasma using the Agilent Bravo Platform. Agilent Application Note 5994-0685EN, 2019.
2. Apffel A, et al. A Novel Solid Phase Extraction Sample Preparation Method for Lipidomic Analysis of Human Plasma. Metabolites. 2021;11(5):294.
3. Funk RS, et al. Metabolomic Profiling to Identify Molecular Biomarkers of Cellular Response to Methotrexate In Vitro. Clin Transl Sci. 2020;13(1):137-146.