Rapid, Label-Free Screening of Diverse Biotransformations by Flow-Injection Mass Spectrometry

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput >= 1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1-50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Automated summary

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography. However, their limited use is due to the requirement of specialised and expensive hardware. We demonstrate that a common platform, single-quadrupole HPLC-MS, can be used for rapid analysis of diverse biotransformations by flow-injection mass spectrometry (FIA-MS), providing validated activity and selectivity information with a one-minute analytical run time per sample. Our results establish FIA-MS as a versatile and reliable alternative to traditional methods for screening enzymatic reactions.