Abstract
Interconnected molecular networks are at the heart of signaling pathways that mediate adaptive plasticity of eukaryotic cells. To gain deeper insights into the underlying molecular mechanisms, a comprehensive and representative analysis demands a deep and parallel coverage of a broad spectrum of molecular species. Therefore, we introduce a simultaneous metabolite, protein, lipid extraction (SIMPLEX) procedure, a novel strategy for the quantitative investigation of lipids, metabolites, and proteins. Compared with unimolecular workflows, SIMPLEX offers a fundamental turn in study design since multiple molecular classes can be accessed in parallel from one sample with equal efficiency and reproducibility. Application of this method in mass-spectrometry-based workflows allowed the simultaneous quantification of 360 lipids, 75 metabolites, and 3327 proteins from 10(6)cells. The versatility of this method is shown in a model system for adipogenesis- peroxisomal proliferator-activated receptor gamma (PPARG) signaling in mesenchymal stem cells-where we utilized SIMPLEX to explore cross-talk within and between all three molecular classes and identified novel potential molecular entry points for interventions, indicating that SIMPLEX provides a superior strategy compared with conventional workflows.