CE–MS Metabolomic and LC–MS Proteomic Analyses of Breast Cancer Exosomes Reveal Alterations in Purine and Carnitine Metabolism

A nanosheath-flow capillary electrophoresis mass spectrometry (CE-MS) system with electrospray ionization was used to profile cationic metabolite cargo in exosomes secreted by nontumorigenic MCF-10A and tumorigenic MDA-MB-231 breast epithelial cells. An in-house-produced sheath liquid interface was developed and machined from PEEK to enable nanoflow volumes. Normalization of CE-MS peak areas to the total UV signal was employed to enhance quantitative accuracy and reduce variability. CE-MS-based metabolomics revealed increased purine synthesis intermediates and increased carnitine synthesis metabolites in MDA-MB-231-derived exosomes, with pathway enrichment indicating the activation of de novo purine pathways and upregulation of carnitine metabolism. In addition, nano-LC-MS-based proteomics revealed differential expression of ecto-5'-nucleotidase (NT5E) and mitochondrial aldehyde dehydrogenase (ALDH9A1), demonstrating metabolic alterations in related enzymatic steps. This study demonstrates the application of nanosheath-flow CE-MS for comprehensive and quantitative exosome metabolomics, uncovering metabolic reprogramming in purine and carnitine pathways between normal and cancerous breast cell lines and providing insight into exosome-mediated signaling of breast cancer metabolism.