Rapid Proteomics to Prospect and Validate Novel Bacterial Metabolism Induced by Environmental Burden.

摘要: Abstract Understanding the pathophysiology of genes and enzymes involved in caffeine metabolism can have extracurricular benefits, such as providing distinct methylxanthines intermediates for pharmaceutical synthesis, also improve environmental waste remediation. The strains Pseudomonas putida CBB5 CES may provide insights into these applications because they both be induced to degrade caffeine, yet latter thrives concentrations > 8.0 g L–1; threefold higher than any other bacteria. We took a novel approach toward identifying enzymatic pathways sp. deletion mutation strain CBB5, which largely circumvented need exhaustive isolation stepwise reconstitution their activities determine response elements. Here, we describe two optimized, rapid alternative strategies based on multiplexed SIL assays demonstrate application by discovering caffeine-degrading quantitative comparison between enriched lysate fractions drawn from bacterial proteomes grown absence presence caffeine. Comparisons were made using stable isotope dimethyl labeling expression differences substantiated reciprocal experiments. role identified proteins degradation was independently verified genetic sequencing. Multiple new components N-demethylase system discovered within fraction specifically this activity. how expand biological context (and reduce systemic bias) adapting protocol total analysis. combined off-line prefractionation with speed resolution advantages Orbitrap LUMOS. global revealed 2406 1789 quantified treatments revealing, among insights, antagonistic pathway vanillin that is completely suppressed treatment.