Systems biology offers a holistic approach to understanding complex biological processes through the integration of quantitative measurements, computational modeling, and simulation. A key requirement for this field is the ability to conduct comprehensive quantitative analyses of multiple molecular layers. This study addresses that need by introducing a mass spectrometry-compatible sample preparation method, enabling the SImultaneous Metabolite, Protein and Lipid Extraction from one sample aliquot (SIMPLEX). The method's versatility was showcased in two distinct model systems: a cell culture model examining peroxisomal proliferator-activated receptor gamma (PPARG) signaling and a more complex biological system, the mouse heart, which was used for an exploratory study of caveolinopathy-associated dilative cardiomyopathy and a focused investigation on myoglobin's role in lipid metabolism. The multi-omics applications demonstrated correlations among the three molecular layers in response to perturbations, highlighting SIMPLEX as an effective strategy for exploring interactions within and between molecular layers in biological systems of varying complexity.
