[4S01a] 09. GteX - Part 5

3:20 PM - 3:40 PM JST(6:20 AM - 6:40 AM UTC)

[4GteX-09] Development of high-throughput metabolic phenotyping using probe electrospray ionization-mass spectrometry

○Nobuyuki Okahashi Okahashi¹ (1. The University of Osaka (Japan))

Keywords:

probe electrospray ionization-mass spectrometry,in vitro enzyme reaction,metabolic pathway prototyping,medium analysis,strain screeing

Metabolic analysis is essential for metabolic engineering tasks such as identifying rate-limiting steps in metabolic pathways and selecting promising strains. In particular, chromatography-based mass spectrometry systems are widely used due to their ability to simultaneously separate and quantify trace metabolites. However, analyses using such systems require labor-intensive sample pretreatment, and chromatographic separation typically takes tens of minutes per sample. Consequently, the analysis of large numbers of samples demands substantial time and effort.In this presentation, we introduce a novel metabolite analysis technique using probe electrospray ionization-mass spectrometry (PESI-MS), which enables rapid measurement with minimal sample preparation. In this method, a small amount of sample (picoliter order) is attached to the tip of a probe and introduced directly into the mass spectrometer. This approach reduces the risk that salts and proteins present in the sample will contaminate the instrument, simplifying the purification process. Sample pretreatment is simple: the sample solution is mixed with an equal volume of ethanol, and the resulting supernatant is directly loaded into the mass spectrometer. In addition, the measurement time per sample is only a few seconds, providing high throughput. Owing to these characteristics, this technique is well suited for the rapid analysis of samples that contain salts and proteins, such as enzyme reaction mixtures, culture media components, and microbial colonies, which are difficult to analyze using conventional chromatography-based mass spectrometry. For example, PESI-MS analysis of in vitro enzyme reaction mixtures prepared with various combinations of enzyme concentrations provides time-course data of metabolite intermediates. From these data, enzyme kinetic parameters such as Km and Vmax can be estimated, enabling rapid identification of reactions for which enzyme concentrations should be optimized to eliminate pathway bottlenecks. Furthermore, direct measurement of microbial culture media using PESI-MS allows rapid identification of by-products secreted into the medium. This approach facilitates the screening of promising host strains and the efficient identification of by-product formation pathways that should be disrupted. In this presentation, we introduce these representative case studies and discuss the potential of high-throughput mass spectrometry for applications in bioproduction.