Multi-Omics for Plasma: A Three-in-One End-to-End Automated Sample Preparation and LC/MS Metabolomics, Lipidomics, and Proteomics Workflow

Importance of the Topic

Integrating metabolomics, lipidomics, and proteomics in a single plasma workflow enables comprehensive pathway mapping, biomarker discovery, and evaluation of therapeutic effects. Automated sample processing combined with targeted triple quadrupole mass spectrometry enhances reproducibility, sensitivity, and throughput, addressing key challenges in large-scale multi-omics studies.

Study Objectives and Overview

This work demonstrates an end-to-end automated workflow for plasma and cell samples using:

• Bravo Metabolomics Sample Prep Platform and AssayMAP Bravo for extraction
• Agilent Infinity II Bio LC (standard flow) or Evosep One (low flow) for separation
• Agilent 6495D triple quadrupole MS for targeted quantitation

The goal is to profile over 500 polar metabolites, 763 lipids across 44 classes, and 375 peptides with high precision and cross-lab transferability.

Methodology and Instrumentation

Sample preparation employs Captiva EMR-Lipid plates on the Bravo platform for metabolites and lipids, and AssayMAP Bravo for proteolytic digestion. Chromatography uses a 16-minute C18 method for lipids and a 24-minute HILIC-Z method for polar metabolites. MRM proteomics kits target 375 mouse plasma peptides. Data acquisition on the 6495D MS achieves dwell times as low as 0.5 ms per transition. Data analysis is performed with MassHunter Quant or Skyline, followed by statistical and pathway interpretation in Mass Profiler Professional with Pathway Architect.

Main Results and Discussion

Automated extraction reduced metabolite variability by approximately 50% compared to manual pipetting, achieving percent relative standard deviations below 2% for key compounds. The lipidomics method reliably quantified 763 species with inter-laboratory RSDs under 20%. Peptide quantification reached lower limits of quantitation below 10 amol on column for nearly half of the targeted peptides. Fine-tuning ion funnel parameters on a per-transition basis improved sensitivity by up to 28% for select metabolites, demonstrating flexibility for analyte-specific optimization.

Benefits and Practical Applications

• High reproducibility and precision across omics modalities
• Attomole-level sensitivity and wide dynamic range
• Reduced hands-on time and enhanced throughput for large cohorts
• Transferable methods ensure consistency across labs

This integrated workflow is well suited for biomarker validation, drug mode-of-action studies, toxicity screening, and precision medicine research.

Future Trends and Applications

Emerging directions include single-cell multi-omics integration, AI-driven real-time data interpretation, expansion of targeted MRM libraries, and further automation in sample handling. Advances in ion funnel design and microflow LC may further boost sensitivity and throughput, opening new possibilities in systems biology and clinical diagnostics.

Conclusion

The described end-to-end automated LC/TQ workflow combines robust sample preparation, high-performance separation, and sensitive detection to deliver a unified platform for plasma multi-omics. Its reproducibility, transferability, and scalability make it a powerful tool for comprehensive pathway analysis and biomarker discovery.

Reference

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7. Van de Bittner GC et al. A Three-in-One End-to-End Automated Sample Preparation and LC/MS Metabolomics, Lipidomics, and Proteomics Workflow for Plasma. ASMS Poster TP 040. 2024.