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

Abstract

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.