Assessing enzyme substrate specificity using combinatorial libraries and electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry

Abstract

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 component of a H-gamma-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.

Effects of kinetic energy and collision gas on measurement of cross sections by Fourier transform ion cyclotron resonance mass spectrometry

“…All experiments were carried out using a Bruker model APEX 47 e Fourier transform ion cyclotron resonance mass spectrometer with an Infinity trapping cell [31,32] and a microelectrospray source modified from an Analytica (Branford, MA) design, with a heated metal capillary drying tube based on the design of Eyler [33]. This instrument has been described in detail in a previous paper [21], so herein just a brief overview is provided.…”

Combinatorial Chemistry Libraries, Analysis of

“…Higger et al 183 monitored the reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and components of a H g Glu Cys Xxx OH (where Xxx is any natural amino acid except Cys, Val, Thr, and Pro) tripeptide library catalyzed by the enzyme glutathione-S-transferase (GST). Assessment of GST substrate specificity relied on detection of the products formed by reaction of the tripeptide library with CDNB, rather than on identification of tight binding ligands to the enzyme.…”

Ultra-high resolution for mass spectrometric analysis of complex and low-abundance mixtures – the emergence of FTICR-MS as an essential analytical tool

“…Finally, FTICR-MS has also been applied in the targetassisted screening of combinatorial mixtures [42,43,44,45,46,47]. These applications make use of the interaction of the library components with molecular targets so that, e.g., the deconvolution of synthetic mixtures is simplified and the screening stage is integrated in the synthesis stage [42].…”

“…These papers are outside the scope of this short review but are nevertheless briefly mentioned. Published work in this field includes studies by Bruce et al [43] on the presence of non-covalent complexes in the gas-phase by FTICR-MS using bio-affinity mass spectrometry (BACMS), by Wigger et al [44] on the reaction of the electrophilic substrate 1-chloro-2,4-dinitrobenzene and components of an H-γ-Glu-Cys-Xxx-OH library catalyzed by glutathione-S-transferase, by Cheng et al [45] and Gao et al [46] on the characterization of non-covalent complexes of proteins with mixtures of ligands, and by Hofstadler et al [47] on the simultaneous measurement of mixtures of aminoglycosides against multiple RNA targets by use of neutral RNA mass tags. In a recent review article Schmid et al [25] give a comprehensive summary of applications of FTICR-MS for screening.…”