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

Importance of the Topic

Multi-omics analysis integrating metabolomics, lipidomics, and proteomics provides a comprehensive view of biochemical changes in biological samples. Such workflows are pivotal for elucidating metabolic pathways, disease mechanisms, and biomarker discovery, especially when working with minimal sample volumes like 20 µL of plasma.

Objectives and Study Overview

This study aimed to develop and validate an automated, end-to-end sample preparation workflow that concurrently extracts metabolites, lipids, and proteins from low-volume plasma samples. The performance was assessed in terms of coverage, reproducibility, and comparability to manual or dual‐omics methods.

Methodology and Instrumentation

The workflow utilized an Agilent Bravo Liquid Handler with VWorks protocols for streamlined extraction, followed by:

• Protein fraction preparation and tryptic digestion on the AssayMap Bravo platform.
• Metabolite and lipid separation using Captiva EMR-Lipid solid phase extraction plates.
• LC/MS analysis:
• Metabolites: HILIC chromatography coupled to a 6495C triple quadrupole.
• Lipids: reversed-phase C18 chromatography on a 6495C triple quadrupole.
• Proteins: reversed-phase chromatography on a 6546 Q-TOF.
• Data processing with commercial quantitative software and Spectrum Mill.

Main Results and Discussion

The automated protocol achieved:

• Detection of 437 metabolites, with 70 % showing total %RSD ≤ 10 % and 87 % ≤ 20 %.
• Identification of 615 lipid species across major classes, with sample preparation %RSD generally below 10 %.
• An average of 1 204 peptide identifications per injection, demonstrating high reproducibility (%RSD ≈ 6 % for peptide counts, median MS1 peak area %CV ≈ 14 %).

Comparisons with manual and two-in-one workflows showed equal or improved performance, consistent chromatographic profiles, and lipid recoveries ranging from 40 % to 120 %.

Benefits and Practical Applications

This three-in-one automated workflow:

• Minimizes hands-on time and operator variability.
• Supports high-throughput processing of limited-volume samples.
• Allows targeted and untargeted analyses on a single LC/MS platform via method switching and wash steps.

Future Trends and Possibilities

Emerging opportunities include:

• Extending the workflow to additional omics layers such as glycomics or polar lipid subclasses.
• Incorporating advanced data analytics and machine learning for integrated multi-omics interpretation.
• Further miniaturization to reduce sample volume requirements.

Conclusion

The automated three-in-one sample preparation reproducibly extracts metabolites, lipids, and proteins from small plasma volumes. Its flexibility, throughput, and ease of implementation accelerate multi-omics studies and biomarker research.

References

1. Vande Bittner GC et al. Automated Dual Metabolite + Lipid Sample Preparation Workflow. Agilent Technical Overview, 2022.
2. Sartain M et al. Automated Low-Volume Plasma Metabolite Extraction. Agilent Application Note, 2020.
3. Spivia W et al. Automated Metabolite Extraction for Plasma. Agilent Application Note, 2019.
4. Apffel A et al. Solid Phase Extraction for Lipidomic Analysis of Human Plasma. Metabolites, 2021.
5. Yannell K et al. End-to-End Targeted Metabolomics Workflow. Agilent Application Note, 2023.
6. Huynh K et al. High-Throughput Plasma Lipidome Analysis. Agilent Application Note, 2021.
7. Wu L. Peptide Quantification in Plasma Using LC/Q-TOF. Agilent Application Note, 2020.