Efficient derivatization-free monitoring of glycosyltransferase reactions via flow injection analysis-mass spectrometry for rapid sugar analytics

The widespread application of enzymes in industrial chemical synthesis requires efficient process control to maintain high
yields and purity. Flow injection analysis-electrospray ionization-mass spectrometry (FIA-ESI–MS) offers a promising solu-
tion for real-time monitoring of these enzymatic processes, particularly when handling challenging compounds like sugars
and glycans, which are difficult to quickly analyze using liquid chromatography-mass spectrometry due to their physical
properties or the requirement for a derivatization step beforehand. This study compares the performance of FIA-MS with
traditional hydrophilic interaction liquid chromatography (HILIC)-ultra high-performance liquid chromatography (UHPLC)-
mass spectrometry (MS) setups for the monitoring of the enzymatic synthesis of N-acetyllactosamine (LacNAc) using
beta-1,4-galactosyltransferase. Our results show that FIA-MS, without prior chromatographic separation or derivatization,
can quickly generate accurate mass spectrometric data within minutes, contrasting with the lengthy separations required
by LC–MS methods. The rapid data acquisition of FIA-MS enables effective real-time monitoring and adjustment of the
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enzymatic reactions. Furthermore, by eliminating the derivatization step, this method offers the possibility of being directly
coupled to a continuously operated reactor, thus providing a rapid on-line methodology for glycan synthesis as well.