Mapping protein-metabolite interactions in E. coli by integrating chromatographic techniques and co-fractionation mass spectrometry

Toward characterization of protein-metabolite interactomes, we recently introduced PROMIS, a co-fractionation-based mass spectrometry approach. However, the challenge lies in distinguishing true interactors from coincidental co-elution when a metabolite co-fractionates with numerous proteins. To address this, we integrated two chromatographic techniques-size exclusion and ion exchange-to enhance the mapping of protein-metabolite interactions (PMIs) in <i>Escherichia coli</i>. This integration aims to refine the PMI network by considering size and charge characteristics, resulting in 994 interactions involving 51 metabolites and 465 proteins. The PMI network is enriched for known and predicted interactions, providing validation. Furthermore, analyzing protein targets for different metabolites revealed functional insights, such as a connection between proteinogenic dipeptides and fatty acid biosynthesis. Notably, we uncovered an inhibitory interaction between the riboflavin degradation product lumichrome and orotate phosphoribosyltransferase, a key enzyme in <i>de novo</i> pyrimidine synthesis affecting biofilm formation. In summary, our integrated chromatographic approach significantly advances PMI mapping.