Single-Sample, Multiomic Mass Spectrometry for Investigating Drug Effects and Mechanisms

Poor therapeutic indexes are a principal cause of drug attrition during development. To develop multiomic methods for elucidating potentially targetable mechanisms of drug toxicity, we performed profiling of the response to subtoxic and toxic doses of l-Asparaginase (ASNase) in immune-compromised mice. ASNase is an enzyme-drug approved for the treatment of pediatric acute lymphoblastic leukemia (ALL) but too toxic for use in adults, making it an ideal test case. We collected 20-μL whole blood samples longitudinally, processed them to plasma, and extracted three molecule types (metabolites, lipids, and proteins) from each sample. We then analyzed the extracts using multiple reaction monitoring (MRM) of 500+ water-soluble metabolites, 750+ lipids, and 375 peptides on a triple quadrupole LC-MS/MS platform. Metabolites, lipids, and peptides that were modulated in a dose-dependent manner appeared to converge on antioxidation, inflammation, autophagy, and cell death pathways, prompting the hypothesis that inhibiting one or more of those pathways might decrease ASNase toxicity while preserving anticancer activity. The present studies were not designed to address therapeutic index directly, because efficacy was not studied. We provide here a streamlined, three-in-one LC-MS/MS workflow for targeted metabolomics, lipidomics, and proteomics and, as a proof of principle, demonstrate its ability to generate new hypotheses about mechanisms of ASNase toxicity.