High-throughput mass-spectrometric enzyme activity determination with PECAN

Assaying for enzymatic activity is a persistent bottleneck in the discovery and engineering of natural product biosynthesis enzymes, and the screening of natural products for desired inhibitory activities. Testing thousands of enzyme mutants (e.g. in directed evolution campaigns) or fractions (e.g. in "grind-and-find" discovery projects) calls for high-throughput enzyme activity assays, but current spectrophotometric assays tend to be applicable only to a narrow range of biochemical transformations. On the other hand, more universal enzyme characterization methods usually require chromatography, greatly diminishing throughput. We have developed a novel enzyme activity assay termed “Probing Enzymes with ‘Click’-Augmented NIMS” (PECAN), which allows for the high-throughput mass-spectrometric detection of enzyme activity in crude cell lysate or whole cell culture, without the need for a chromatographic step. We demonstrate the applicability of this technology to the rapid determination of acetyltransferase and cytochrome P450 activity in both lysate and whole cells.