Lipid Profiling Workflow Demonstrates Disrupted Lipogenesis Induced with Drug Treatment in Leukemia Cells

Importance of the topic

Lipidomics profiling provides a comprehensive view of cellular lipid composition and dynamics. In cancer research and drug development, detailed lipid analysis reveals pathway dysregulation, biomarker candidates, and mechanisms of drug action. High-resolution liquid chromatography coupled to time-of-flight mass spectrometry enables deep coverage of complex lipidomes, supporting both targeted and untargeted discovery.

Objectives and Study Overview

• Investigate lipidome changes in acute myeloid leukemia K562 cells treated with bezafibrate, medroxyprogesterone acetate, and their combination (BaP).
• Apply the Agilent 6546 LC/Q-TOF and MassHunter Lipid Annotator software for iterative MS/MS and deep lipid annotation.
• Compare results to prior studies and expand the set of identified lipid classes and species.

Methodology

Four biological replicates per treatment were incubated for 24 hours, pelleted, washed and flash-frozen. Lipids were extracted using a modified Folch procedure. Extracts were split for positive and negative ion analysis. Targeted MS1 data and AutoMS/MS iterative fragmentation were acquired. Untargeted workflows employed recursive feature extraction. Data processing included Profinder for feature finding, Lipid Annotator for annotation, Mass Profiler Professional for statistical analysis and ID Browser for untargeted annotation.

Instrumentation

• LC: Agilent 1290 Infinity II with High Speed Pump, Vialsampler and Multicolumn Thermostat.
• Column: Agilent Poroshell 120 EC-C18 3.0×100 mm, 2.7 µm, with matching guard.
• MS: Agilent 6546 LC/Q-TOF with Jet Stream ion source.
• Software: MassHunter Q-TOF Acquisition v10, Lipid Annotator v1.0, PCDL Manager B.08 SP1, Profinder v10.0, Mass Profiler Professional v15.1, ID Browser v10.0.

Main Results and Discussion

Multivariate analysis (PCA, hierarchical clustering) clearly separated treatment groups. BaP led to a marked increase in triacylglycerols and decrease in diacylglycerols, consistent with disrupted de novo lipogenesis. Additional changes included elevated ceramide NS and hexosylceramide NS levels and reduced phosphatidylcholine and cardiolipin classes. Chromatographic separation resolved lipid isomers such as Cer_NS d18:1_24:1 and d18:2_24:0 with opposite responses to treatment. Untargeted analysis uncovered a novel C2 ceramide (N-acetylsphingosine) with a 3.9-fold increase in BaP cells, suggesting a role in drug-induced apoptosis.

Benefits and Practical Applications

This comprehensive workflow enhances lipid annotation coverage and supports mechanistic studies of drug effects, biomarker discovery, QA/QC in pharmaceutical and clinical research. The ability to resolve isomers and detect low-abundance lipids expands understanding of lipid mediated signaling and metabolism.

Future Trends and Potential Applications

Future directions include integration with single-cell lipidomics, imaging mass spectrometry and multiomic data fusion. Expanded in silico libraries and AI-assisted annotation will accelerate discovery. Clinical translation for precision medicine and routine QA/QC applications will benefit from further automation and throughput increases.

Conclusion

The Agilent lipidomics profiling workflow combining the 6546 LC/Q-TOF and MassHunter Lipid Annotator delivers robust targeted and untargeted lipid analysis. Application to AML cells under lipid-modulating drug treatments confirmed known effects and revealed novel lipid class changes and an atypical ceramide. This approach supports in-depth mechanistic studies and advances in translational lipidomics.

References

• Southam AD et al Drug Redeployment to Kill Leukemia and Lymphoma Cells by Disrupting SCD1 Mediated Synthesis of Monounsaturated Fatty Acids Cancer Res 2015 75 12 2530 2540
• Sartain M et al Improving Coverage of the Plasma Lipidome Using Iterative MS/MS Data Acquisition Combined with Lipid Annotator and 6546 LC Q TOF Agilent Technologies Application Note 5991 0775EN 2019
• Agilent MassHunter Profinder Batch Processing for High Quality Feature Extraction of Mass Spectrometry Data Technical Overview 5991 3947EN 2014
• Agilent Lipidomics Analysis with Lipid Annotator and Mass Profiler Professional Technical Overview 5994 1111EN 2019
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