Optimizing multiomics sample preparation: comparative evaluation of extraction protocols for HepG2 cells

Multiomics approaches enable a comprehensive characterization of complex biological systems by simultaneously investigating multiple molecular layers. Generating multiple omics datasets from a single sample is crucial to minimize biological variability and ensure cross-layer consistency, which is critical for robust downstream data analysis. However, existing workflows often require adaptation to the specific experimental context and instrumental setup. This study systematically compared two established protocols for the simultaneous extraction of metabolites, lipids, and proteins from HepG2 cells: (i) a biphasic extraction with subsequent overnight protein digestion from the interphase pellet, and (ii) a monophasic extraction involving on-bead protein digestion. For the monophasic approach, we further investigated the effects of bead size and digestion conditions. Metabolomics samples were analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry; lipidomics and proteomics samples were analyzed by nano-scale liquid chromatography coupled with ion mobility separation and high-resolution tandem mass spectrometry. Each method was evaluated in terms of total feature count, selectivity, reproducibility, handling complexity, and overall performance. While neither protocol was optimal across all criteria, the monophasic extraction using paramagnetic beads with shortened incubation time proved to be the most reproducible, efficient, and cost-effective solution for in-house multiomics workflows in HepG2 cells.