Assessing Enzyme Substrate Specificity Using Combinatorial Libraries and Electrospray Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Maria Wigger 1,2 , Joseph P. Nawrocki 1 , Clifford H. Watson 1 , John R. Eyler 1 * and Steven A. Benner 1 1 Department of Chemistry, P.O. Box 117200, University of Florida, Gainesville, FL 32611-7200, USA 2 Department of Chemistry, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland SPONSOR REFEREE: J. Amster, Department of Chemistry, University of Georgia, Athens, GA 30602-2556, USA A model experiment for the ‘on-line’ screening of substrate libraries by enzymes using combinatorial libraries in combination with electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry has been performed. The reaction between the electrophilic substrate 1-chloro-2,4-dinitrobenzene and components of a H-γ-Glu-Cys-Xxx-OH library, catalyzed by glutathione-S-transferase, has been monitored. It shows the feasibility of ‘two-dimensional’ screening of substrate libraries by ESI-FTICR mass spectrometry. © 1997 John Wiley & Sons, Ltd. Received 2 September 1997; Accepted 2 September 1997 Rapid. Commun. Mass Spectrom. 11, 1749–1752 (1997) No. of Figures: 3 No. of Tables: 0 No. of Refs: 21 Determining the substrate specificity of enzymes in biological systems is a major challenge in biological research, important in biotechnology, the design of drugs, and the assessment of physiological roles in biological systems. 1 Emerging combinatorial technol- ogy offers a powerful way to address this challenge. Although peptide, 2 oligonucleotide 3 and various small molecule 4–7 libraries have been successfully used to explore receptor–ligand interactions, 8,9 very few studies have been performed where components of libraries are enzymatically transformed as part of the combina- torial experiment. 10 This situation challenges the field of analytical chemistry to develop instrumentation for the high throughput screening of complex libraries. In response to these challenges, applications for sophisti- cated mass spectrometric instrumentation have been established that allow rapid, sensitive and high through- put analysis of complex libraries. 11,12 Glutathione-S-transferases (GST) are intracellular, heterodimeric enzymes that play a crucial role in the detoxification of alkylating agents in the cell. 13,14 These enzymes catalyze the nucleophilic attack of glutathio- ne(g-glutamyl-cysteinyl-glycine) on a wide variety of hydrophobic electrophiles, including drugs and envi- ronmental pollutants, yielding conjugated or trans- formed metabolites that are less toxic and more easily excretable. Several electrophiles are used in in vitro assays as arbitrary electrophilic substrates to determine the activity of GSTs. 15 Consistent with their physiological role, the struc- tures of the electrophiles that GST accepts are quite diverse. Nevertheless, different isozymes of GST exist with different specificities for the electrophile. This is a common strategy in evolving a spectrum of detoxifying enzymes in higher organisms. Mammalian liver, for example, contains a set of isozymes of alcohol dehy- drogenases that serve as detoxification enzymes; each is quite broad in terms of specificity, but with the collection of specificities presumably engineered to cover the spectrum of possible xenobiotic substances that might challenge the organism. The assessment of substrate specificity of enzymes by combinatorial libraries offers a new alternative for the fast discovery of natural substrates and inhibitors. We report here the first example of the use of a powerful high resolution mass spectrometric method, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR), 16 for determining substrate specificity in a ‘two-dimensional’ combinatorial experiment involving GST. We report that this technology permits combinatorial substrate libraries to be screened for substrate specificity in a simple experiment without tedious workup. EXPERIMENTAL The experiments were performed at the University of Florida on a Bruker 47e external source FTICR mass spectrometer (Bruker Instruments Inc., Billerica, MA, USA) equipped with a shielded, horizontal, room- temperature, 15 cm inside diameter, 4.7 T super- conducting magnet (Magnex Scientific Ltd., Abingdon, England). Ions produced externally to the magnet were guided into the 170 mm 3 cylindrical RF-shimmed Infinity™ analyzer cell 17 using electrostatic ion optics. The standard external source FTICR instrument has been previously described 18 and only relevant details are discussed further. The FTICR mass spectrometer was equipped with an external electrospray source which utilizes a hexapole ion guide (Analytica of Branford, Branford, CT, USA). The ESI source was modified in the laboratory to use a heated metal *Correspondence to: J. R. Eyler, Department of Chemistry, P.O. Box 117200, University of Florida, Gainesville, FL 32611-7200, USA Contract/grant sponsor: National Science Foundation Contract/grant sponsor: Swiss Federal Government Contract/grant sponsor: National High Magnetic Field Laboratory In-House Research Program RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 11, 1749–1752 (1997) CCC 0951–4198/97/161749–04 $17.50 © 1997 John Wiley & Sons, Ltd.