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 Ludovic T. Maillard, Vincent Gue ´ rineau, Marie-Ange Badet-Denisot, Bernard Badet, Olivier Lapre ´ vote * and Philippe Durand Institut de Chimie des Substances Naturelles, UPR 2301-CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France Received 15 November 2005; Revised 20 December 2005; Accepted 24 December 2005 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 glucosa- mine-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-( 13 C 2 )acetylglucosamine-6P as internal standard. Since no isotopically substituted GlcN-6P was available, the N-( 13 C 2 )acetyl derivative, easily obtained from ( 13 C 4 )-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. Copyright # 2006 John Wiley & Sons, Ltd. The potential of mass spectrometry (MS) for the direct analysis of complex biological samples has been extensively demonstrated. In particular, electrospray ionization (ESI)- MS has been shown to be particularly useful for the study of enzyme kinetics, and has become an excellent technique complementary to photometric, fluorometric and radio- metric assays (for a review, see 1,2 ). However, since ESI-MS has low tolerance toward sample contaminants, especially salts, a preliminary or combined separation by high- performance liquid chromatography (HPLC) is usually required. Quantification with matrix-assisted laser de- sorption/ionization time-of-flight (MALDI-TOF) MS, which is rapid and very sensitive, has been limited in quantitative analysis due to significant variations in ion signals. Several parameters must be taken into account such as shot-to-shot reproducibility, homogeneity of the matrix and sample co- crystallization, signal suppression effects, fluctuation in laser power and non-linear detector response. Introduction of an internal standard minimizes these problems. The best internal standard for MALDI-TOFMS quantification is a stable isotope containing an analog of the compound to quantify that is distinguishable by mass analysis but with no change in the chemical properties. 3 The main advantage is that the laser desorption and ionization rates are the same for both compounds. A number of reports have described the use of MALDI- TOFMS to follow the time course of enzyme reactions. Kang et al. 3,4 reported the quantitative determination of substrates and products of an immobilized lipase-catalyzed reaction in non-buffered organic solvent. More recently, Bungert et al. 5 investigated the conversion of D-glucose into D-gluconic acid catalyzed by glucose oxidase. Chemical screening of enzyme inhibitors has also been reported using the SAMDI (self-assembled monolayers for MALDI) method. 6 The present work reports the quantification of D-glucosamine- 6-phosphate using N-( 13 C 2 )acetylglucosamine-6P as internal standard by MALDI-TOFMS analysis and in situ N- acetylation. Its use for the kinetic study of glucosamine-6P synthase (Glms) and some inhibition assays is also described. Glucosamine-6-phosphate synthase (Glms, Gfa or Gfat) 7,8 catalyzes the formation of D-glucosamine-6P (GlcN-6P) and L-glutamate from D-fructose-6P (Fru-6P) and L-glutamine. This is the first and rate-limiting step of the hexosamine biosynthetic pathway (Eqn. (1)). Its involvement in the hyperglycemia-induced malfunction of cells 9 makes it a potential medicinal target to prevent complications of type II diabetes. 10,11 D-fructose-6P þ L-glutamine ! ÀÀ D-glucosamine-6P þ L-glutamate (1) Assays for Glms activity have been reported based either on the quantification of the L-glutamate or the GlcN-6P. Determination of L-glutamate was realized by spectropho- tometric or fluorometric glutamate-dehydrogenase coupled assays, 12–14 but has some limitations for the testing of inhibi- tors. 15 Monitoring of GlcN-6P formation seems more relevant RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 666–672 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2361 *Correspondence to: O. Lapre ´vote, Institut de Chimie des Sub- stances Naturelles, UPR 2301-CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France. E-mail: olivier.laprevote@icsn.cnrs-gif.fr Contract/grant sponsor: Institut de Chimie des Substances Naturelles. Copyright # 2006 John Wiley & Sons, Ltd.