Unknown metabolite identification remains a major challenge. In this presentation, I will discuss how stable isotope labeling can facilitate untargeted analysis as well as identifying unknown metabolites from microbial samples. First, analysis of unlabeled and labeled sample facilitate accurate LC-MS peak categorization. Biological peaks can be distinguished from nonbiological peaks based on whether the peaks exhibit mass shifts between unlabeled and labeled samples. Moreover, the mass shifts can be used to derive C and N atom counts, with carbon or nitrogen atoms coming from adducting remaining unlabeled. Adduct and metabolite peaks share sane C and N counts, facilitate their annotation. Secondly, the C and N counts determined from labeling patterns help to find correct formula when multiple formulae match the measured m/z. Thirdly, isotope labeling can be used to resolve structural isomers for a single formula. Examples will be given for the determination of correct isomer with a formula of C5H13O8P associated with yeast gene YNL010W through an elegant pulse chase experiment. Finally, isotope labeling helps to resolve certain experimental artifacts from true metabolites. For example, we observe that some biological peaks have C and N count not matching database formula even though the m/z are perfectly matched. We show that some of these unknown compounds result from reaction between endogenous metabolites and formate in extraction buffer, with the carbon atom from formate remaining unlabeled. These “reaction products” can be mistaken as metabolites.

Metabolite discovery from isotope labeling

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.