A High-Throughput Lipodomic Workflow Using the Waters Xevo MRT Mass Spectrometer

Importance of Topic

Lipidomics plays a crucial role in understanding disease mechanisms and identifying biomarkers through comprehensive profiling of lipids in biological samples. High-throughput approaches are essential for large-cohort studies in biomedical and epidemiological research, enabling reproducible and accurate lipid measurement across hundreds of samples. The described workflow addresses these needs by combining advanced separation techniques, high-resolution mass spectrometry, and dedicated informatics tools.

Objectives and Study Overview

This study presents a high-throughput lipidomic workflow using the Waters Xevo MRT Mass Spectrometer to analyze human serum samples from colorectal cancer (CRC) patients and healthy controls. The key aims are:

• To evaluate instrument stability, mass accuracy, and resolution over extended sample runs.
• To achieve confident identification and quantification of lipid species in a large sample set.
• To integrate chromatographic separation, MS acquisition, and data processing for rapid profiling.

Methodology and Instrumentation

The workflow combines ultra-performance liquid chromatography with multi-reflecting ToF mass spectrometry:

• Sample Set: 12 human plasma/serum samples, including CRC and control cohorts, diluted with EquiSPLASH internal standard.
• LC Conditions:
  Flow rate: 0.4 mL/min
  Column: UPLC CSH C18 2.1×50 mm, 1.7 μm at 55 °C
  Mobile Phases: A) Water/IPA/MeCN (50:25:25) with 5 mM ammonium acetate and 0.05% acetic acid
  B) IPA/MeCN (50:50) with 5 mM ammonium acetate and 0.05% acetic acid
  Gradient: 4-minute rapid gradient with initial hold, organic ramp, and re-equilibration.
• MS Conditions:
  Instrument: Xevo MRT Mass Spectrometer
  Ionization: ESI positive/negative
  Mass Range: m/z 50–1200
  Acquisition Rate: 1, 5, 10, 20 Hz (up to 100 Hz in MS/MS)
  Resolution: 100,000 FWHM at 100 Hz
  Acquisition Mode: MSE with collision energy ramp 20–45 eV
  Source: Desolvation at 500 °C; Cone voltage 40 V.
• Data Processing: waters_connect applications to convert to mzML; LipoStar2 for lipid identification, statistical analysis, and pathway profiling.

Key Results and Discussion

• Instrument Performance:
  Mass accuracy maintained below 1 ppm RSD over 200+ injections.
  Retention time precision of 0.1% RSD and peak area variation of ≤8% RSD for deuterated standards.
• Resolution Consistency:
  Achieved ~80,000 FWHM across scan rates from 1 to 20 Hz, demonstrating robust multi-reflecting ToF performance.
• Biomarker Identification:
  Differential lipid signature of ceramide Cer(18:1/24:0) upregulated in CRC versus control samples via pathway analysis.
  High data quality allowed use of tight mass and retention time tolerances for rapid compound annotation.
• Throughput and Reproducibility:
  Rapid 4-minute LC/MS cycle enabled over 200 injections with consistent data, suitable for large-cohort studies.

Benefits and Practical Applications

• High-throughput capacity supports large-scale clinical and epidemiological projects.
• Exceptional mass accuracy and resolution enable confident lipid identification and quantitation.
• Integration of chromatography, MS, and informatics streamlines workflow and reduces turnaround time.
• Applicable to disease biomarker discovery, drug development, and quality control in lipid analysis.

Future Trends and Possibilities

• Expansion to higher acquisition rates (up to 50–100 Hz) for faster throughput without compromising resolution.
• Integration with ion mobility for enhanced structural characterization of lipid isomers.
• Advanced machine learning approaches in data processing for automated lipid annotation and quantitation.
• Broader application in multi-omics studies by coupling lipidomics with proteomics and metabolomics data.

Conclusion

The described lipidomic workflow leveraging the Waters Xevo MRT Mass Spectrometer demonstrates rapid, high-resolution analysis with outstanding reproducibility. It provides a robust platform for large-cohort lipid profiling, facilitating biomarker discovery and mechanistic insights in biomedical research.

Reference

• Morgan et al., Gut 2023;72:338-344. doi:10.1136/gutjnl-2022-327736
• King A., Gethings L.A., Plumb R.S. Waters Application Note 720006321, 2018.
• Goracci L. et al., Anal. Chem. 2017, 89(11):6257–6264
• Gomez‐Larrauri A. et al., Medicina 2021, 57(7):729