C.L. Yu, S. Brooks, Y. Li, M. Subramanian, R. Summers, M. Pope

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