Abstract
Understanding the dynamic cellular metabolism is essential for gaining deeper insights into inter- and intracellular functions. In recent years, mass spectrometry (MS) has become the technology of choice for the biochemical characterization and profiling of cell lines, particularly when coupled with separation techniques such as liquid chromatography (LC-MS). However, these methods typically involve extensive sample preparation with potent organic solvents, which is labor-intensive, time-consuming, and incompatible with direct analysis of intact, live cells. Here, we propose the use of the ambient ionization technique Laser Desorption-Rapid Evaporative Ionization Mass Spectrometry (LD-REIMS) incorporated in an automated platform, for the high-throughput profiling of live or frozen cell monolayers, with minimal pretreatment. Validation experiments using 10 breast and colorectal cancer cell lines confirmed high accuracy, repeatability, and molecular coverage of the method, with over 400 metabolites and lipids detected and identified, including saccharides, amino acids, fatty acids, and glycerophospholipids. Of these, 144 were further confirmed and quantified with LC-MS/MS and standard compounds. We also applied the method to establish lipidomic differences across the isogenic MCF10A cells harboring either WT or MUT PIK3CA. Finally, we conducted time-series experiments on hypoxic cells, which revealed significant dynamic changes in metabolism, including lactate accumulation due to anaerobic glycolysis.