Monitoring enzyme‐catalyzed production of glucosamine‐6P by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: a new enzymatic assay for glucosamine‐6P synthase

Abstract

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) method for quantification of D-glucosamine-6P (GlcN-6P) that allows the kinetic study of glucosamine-6P synthase (Glms) is presented. The present report describes the optimization of the different steps of a new enzymatic assay for Glms based on in situ N-acetylation of GlcN-6P and MALDI-TOFMS analysis using N-(13C2)acetylglucosamine-6P as internal standard. Since no isotopically substituted GlcN-6P was available, the N-(13C2)acetyl derivative, easily obtained from (13C4)-acetic anhydride, was used as internal standard. Validation of the assay was achieved by measuring the fructose-6P Michaelis constant, in full agreement with reported values, and by studying the inhibition properties of arabinose-5P oxime.

Discovery of Rifampicin as a New Anti-Glycating Compound by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry-Based Insulin Glycation Assay

“…Earlier studies have shown that MALDI-tof-MS can be used for quantitative purposes, for example, it was used for the determination of substrates and products of an immobilized lipase-catalyzed reaction in non-buffered organic solvent. 11 Similarly, the conversion of D-fructose into glucosamine-6P catalyzed by glucosamine-6P synthase was analyzed by MALDI-MS. 12 In our previous studies, we have used MALDItof-MS for the quantification of cystatin, 13 as well as to quantify the inhibition of glycation of low abundant proteins by albumin. 14 In this study, we describe a high-throughput MALDI-tof-MS-based in vitro insulin glycation assay for screening glycation inhibitors.…”

The use of matrix‐assisted laser desorption/ionization mass spectrometry in enzyme activity assays and its position in the context of other available methods

“…The reactions occurred in a non‐disturbing diluted Tris buffer and the N ‐acetylation was necessary to better distinguish glucosamine phosphate from fructose phosphate of a similar mass. For measuring acetylated glucosamine phosphate alone, the authors used a THAP matrix (Maillard et al, 2006) whereas 9‐aminoacridine allowed to measure N ‐acetylated glutamate and N ‐acetylglucosamine‐6‐phosphate simultaneously in the negative mode (Gaucher‐Wieczorek et al, 2014). The determined kinetic constants K m and k cat for the two substrates were comparable with data from other methods.…”

Mass spectrometry for enzyme assays and inhibitor screening: An emerging application in pharmaceutical research

“…Using this approach, they achieved good linear relationships and produced kinetic values in line with those reported from other methods. Similarly, Maillard et al (2006) developed a MALDI‐TOF‐based assay to evaluate the kinetic parameters of glucosamine‐6P synthase. They validated the method by determining the Km for fructose‐6P and with a known competitive inhibitor.…”