Direct Microplate Sampling Mass Spectrometry for High-Throughput Screening of Biocatalytic Activity

The development of biocatalysis depends heavily on high-throughput screening (HTS) approaches to uncover engineered enzymes with superior biocatalytic activity. Recent advances in mass spectrometry (MS) enable label-free, high-speed analysis of biocatalytic samples in standard microplates by omitting the chromatographic separation. However, most MS methods need a complicated sampling interface system and face challenges due to biological matrix interferences, which can diminish the sensitivity and reliability. Herein, we established a direct microplate sampling (DMS)-MS technique for HTS of the enzymatic activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) in steroid biocatalysis. The implementation of an open port interface (OPI) probe with a customized ion source for microwell direct insertion sampling and ionization has streamlined the setup, reduced operational complexity, and shortened the analysis time to 0.9-6.5 s/sample. A sample preparation strategy involving derivatization, followed by high-ratio dilution (e.g., 10,000-fold), was further integrated prior to DMS-MS for mitigating ion suppression effects across diverse biological matrices. Ultimately, this workflow, when applied for monitoring multiple steroids in 17β-HSD biocatalytic processes, demonstrated high stability (peak area RSDs < 7%), minimal carryover (<1%), and quantitative accuracy comparable to conventional liquid chromatography (LC) experiments while providing a >150-fold throughput enhancement. After screening a total of 5760 17β-HSD mutants across three different steroid conversion reactions, we identified and verified two new variants with enhanced biocatalytic activities. This study successfully established a DMS-MS-based HTS workflow for microplate screening, providing a simplified, robust, and highly reliable analytical platform for microbial or enzyme engineering projects involving complex biological matrices.