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Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics
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Optimizing multiomics sample preparation: comparative evaluation of extraction protocols for HepG2 cells.

Tilman F Arnst1, Selina Hemmer1, Claudia Fecher-Trost1

  • 1Department of Experimental and Clinical Toxicology and Pharmacology, Institute of Experimental and Clinical Pharmacology and Toxicology, Center for Molecular Signaling (PZMS), PharmaScienceHub (PSH), Saarland University, Homburg, Germany.

Analytical and Bioanalytical Chemistry
|November 19, 2025
PubMed
Summary
This summary is machine-generated.

This study compared two multiomics extraction methods for HepG2 cells. Monophasic extraction using paramagnetic beads offered the most reproducible, efficient, and cost-effective approach for simultaneous metabolite, lipid, and protein analysis.

Keywords:
HepG2 cellsMass spectrometryMetabolomicsMultiomicsProteomicsSample preparation

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Area of Science:

  • Biochemistry
  • Proteomics
  • Metabolomics
  • Lipidomics

Background:

  • Multiomics approaches integrate data from multiple molecular layers for comprehensive biological system characterization.
  • Simultaneous generation of multiomics data from single samples is vital for minimizing biological variability and ensuring cross-layer consistency.
  • Existing multiomics workflows often require significant adaptation to specific experimental conditions and instrumentation.

Purpose of the Study:

  • To systematically compare two established protocols for simultaneous metabolite, lipid, and protein extraction from HepG2 cells.
  • To evaluate the impact of bead size and digestion conditions within a monophasic extraction workflow.
  • To identify the most reproducible, efficient, and cost-effective multiomics extraction protocol for HepG2 cell line applications.

Main Methods:

  • Comparison of a biphasic extraction protocol (interphase pellet digestion) versus a monophasic extraction protocol (on-bead digestion).
  • Optimization of monophasic extraction by investigating bead size and digestion conditions.
  • Analysis of metabolomics, lipidomics, and proteomics data using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS) and ion mobility separation.

Main Results:

  • Both biphasic and monophasic protocols were evaluated based on feature count, selectivity, reproducibility, handling complexity, and overall performance.
  • The monophasic extraction using paramagnetic beads with a shortened incubation time demonstrated superior reproducibility.
  • The optimized monophasic approach was found to be more efficient and cost-effective compared to the biphasic method.

Conclusions:

  • Neither extraction protocol was universally optimal across all evaluated criteria.
  • The monophasic extraction utilizing paramagnetic beads with optimized digestion conditions presents the most advantageous solution for in-house multiomics workflows in HepG2 cells.
  • This optimized protocol enhances reproducibility, efficiency, and cost-effectiveness for integrated omics data generation.