Issue 14, 2017

Chemoselective ligation reaction of N-acetylglucosamine (NAG) with hydrazide functional probes to determine galactosyltransferase activity by MALDI mass spectrometry

Abstract

Quantification of β-1,4-galactosyltransferase (β-1,4-GT) activity is of considerable significance in the diagnosis of various cancers including lung and ovarian cancer. We report here the use of synthetic β-N-acetylglucosamine (NAG) ligands that contain hydrazide functional groups to determine galactosyltransferase activity by mass spectrometry. With hydrazide-linked β-D-NAG as the acceptor, the activity of β-1,4-GT is quantified by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) with high efficiency. Using the disulfide moiety in a 3,3′-dithiodipropionic acid dihydrazide (DTP)-linked β-D-NAG probe, Au nanoparticles (AuNPs) are employed for enriching DTP-linked β-D-NAG after enzymatic reaction, and the ligand-bound AuNPs are subsequently deposited on a MALDI plate for analysis. In addition, we have demonstrated that a perfluorocarbon (PF) labeled β-NAG-ligand can be useful for surface-based enzymatic reaction with a perfluorooctadecanethiol (PFDT)-covered gold surface. Using the ratiometric method, the conversion rate of β-1,4-GT is determined to be 0.83 ± 0.03, which shows a high level of activity. This is the first work that uses hydrazide-linked β-D-NAG for activity analysis, providing a new surface-based MS approach to determine enzyme activity in a potentially high-throughput manner.

Graphical abstract: Chemoselective ligation reaction of N-acetylglucosamine (NAG) with hydrazide functional probes to determine galactosyltransferase activity by MALDI mass spectrometry

Supplementary files

Article information

Article type
Paper
Submitted
10 Mar 2017
Accepted
14 May 2017
First published
17 May 2017

Analyst, 2017,142, 2654-2662

Chemoselective ligation reaction of N-acetylglucosamine (NAG) with hydrazide functional probes to determine galactosyltransferase activity by MALDI mass spectrometry

H. Yang and Q. Cheng, Analyst, 2017, 142, 2654 DOI: 10.1039/C7AN00428A

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