An LC/MS Metabolomics Discovery Workflow for Malaria-Infected Red Blood Cells Using Mass Profiler Professional Software and LC-Triple Quadrupole MRM Confirmation

Significance of the topic

Malaria infection triggers complex metabolic changes in red blood cells that are critical to understand disease mechanisms and identify therapeutic targets.
Combining untargeted LC/MS profiling with targeted LC/MS/MS confirmation enables both broad discovery and precise quantification.

Objectives and study overview

Develop and demonstrate a combined discovery and confirmation metabolomics workflow.
Compare metabolite profiles of non-infected (NRBC) and infected red blood cells (IRBC) to identify alterations in arginine metabolism.
Utilize Agilent Mass Profiler Professional software and LC-triple quadrupole MRM assays for data mining and confirmation.

Methodology and workflow

• Sample Preparation and Extraction: Cultured Plasmodium falciparum-infected and control erythrocytes were extracted at pH 2, 7 and 9 using biphasic methanol/water/chloroform. Filtration through 0.2 µm and 10 kDa filters produced protein-free extracts.
• Discovery LC/Q-TOF Analysis: Reverse phase chromatography on Agilent 1200 RRLC system coupled to 6520 Accurate-Mass Q-TOF. Data extraction via Molecular Feature Extraction (MFE) and Find by Formula (FbF) in MassHunter Qual. Statistical analysis, PCA, volcano plots and compound identification in Mass Profiler Professional with METLIN database.
• Targeted LC/QQQ Confirmation: Aqueous normal phase separation on Cogent Microsolv Diamond Hydride column with Agilent 6460 Triple Quadrupole LC/MS and Jet Stream. MRM assays for arginine, citrulline and ornithine with calibration over five orders of magnitude for quantitation.

Instrumentation

• Agilent 1200 Series Rapid Resolution LC (binary pump, vacuum degasser, thermostatted autosampler and column compartment)
• Agilent 6520 Accurate-Mass Q-TOF LC/MS system (dual ESI source)
• Agilent 6460 Triple Quadrupole LC/MS system with Jet Stream technology
• Cogent Microsolv Diamond Hydride and ZORBAX reversed phase columns

Main results and discussion

• Thousands of molecular features detected across pH conditions; PCA separated samples by extraction pH and infection status.
• Volcano plots identified arginine (m/z 175.1190) as significantly reduced or absent in IRBC versus NRBC (16-fold decrease).
• Citrulline and ornithine were elevated in infected cells, indicating perturbation of the arginine biosynthesis pathway.
• Hierarchical clustering confirmed distinct metabolic profiles between IRBC and NRBC.
• Pathway analysis highlighted the arginine metabolism network and guided targeted extraction of pathway intermediates.
• MRM quantification validated the discovery findings with an eightfold reduction in arginine and increased citrulline and ornithine levels in IRBC.

Benefits and practical applications

The combined untargeted-targeted workflow offers comprehensive metabolome coverage and high sensitivity.
Integrated software tools streamline statistical analysis, compound identification and pathway visualization.
This approach supports biomarker discovery, drug target validation and quality control in clinical or industrial metabolomics.

Future trends and opportunities

• Integration with multi-omics and machine learning for deeper biological insights.
• Standardization of protocols and expansion of spectral databases to improve compound coverage.
• Advances in high-resolution and multiplexed MS technologies for higher throughput and sensitivity.
• Application to larger clinical cohorts for robust biomarker validation and precision medicine.

Conclusion

A dual-mode LC/MS strategy effectively uncovers and confirms infection-related metabolic perturbations in red blood cells.
The workflow demonstrates a powerful combination of global discovery with targeted quantitation and pathway context.

References

1. T. R. Sana, K. Waddell, S. M. Fischer, “A sample extraction and chromatographic strategy for increasing LC/MS detection coverage of the erythrocyte metabolome,” Journal of Chromatography B 871:314–321 (2008).