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

• Published in: Analytical and bioanalytical chemistry 2024-09, Vol.416 (23), p.5191-5203
• Main Authors: Thiele, Ulrich, Crocoll, Chantal, Tschöpe, André, Drayß, Carla, Kirschhöfer, Frank, Nusser, Michael, Brenner-Weiß, Gerald, Franzreb, Matthias, Bleher, Katharina
• Format: Article
• Language: English
• Subjects: Amino Sugars - analysis; Analytical Chemistry; Biochemistry; Characterization and Evaluation of Materials; Chemical reactions; Chemical synthesis; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, High Pressure Liquid - methods; Data acquisition; Enzymatic synthesis; Flow injection analysis; Flow Injection Analysis - methods; Food Science; Glycan; Glycosyltransferase; Glycosyltransferases - metabolism; High performance liquid chromatography; Injection; Ionization; Laboratory Medicine; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Monitoring; Monitoring/Environmental Analysis; N-Acetyllactosamine; N-Acetyllactosamine synthase; Physical properties; Polysaccharides; Process control; Process controls; Real time; Research Paper; Scientific imaging; Spectrometry, Mass, Electrospray Ionization - methods; Sugar
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-024-05457-9

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 solution 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 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. Graphical Abstract