Karsten Hiller - Shedding light on the unexpected - stable-isotope assisted non-targeted metabolic profiling

Current metabolomics techniques provide only a static view on metabolite amounts present inside or outside of living cells. Stable-isotope labeled tracers can be applied to obtain dynamic information on biochemical reaction networks. However, such methods like for example Metabolic Flux Analysis (MFA) only provide information on known compounds in a targeted manner and rely on extensive a priori knowledge. To shed light on yet unknown parts of the metabolism, we developed the Non-targeted Tracer Fate Detection (NTFD) methodology to detect all known and unknown compounds derived from a stable-isotope labeled tracer present in a GC/MS chromatogram. For every detected and labeled compound mass isotopomer distributions (MIDs) are determined. MIDs of known and unknown compounds are then used as input for the Mass Isotopolome Analyzer (MIA). Based on the non-targeted labeling data, this software reveals global flux changes in known and unknown parts of the metabolic network. During my presentation I will introduce these tools and provide examples on how we applied those to address biological problems in our research and highlight unexpected findings: 1. I will report about a new metabolic driven antimicrobial defense mechanism in macrophage and microglial cells. By using non-targeted metabolomics, we identified a metabolic pathway that these immune cells apply to produce high amounts of an antimicrobial metabolite. 2. By the application of MIA to analyze GC/MS data of stable-isotopic labeled cell extracts of lung cancer cells, we found the neuronal compound N-acetyl-aspartate (NAA) and a neuro-peptide in these cells. We could show that NAA aspartate is involved in fatty acid synthesis and that it is important for fast proliferation.