CE‐ESI‐MS/MS as a rapid screening tool for the comparison of protein–ligand interactions

Abstract

In drug development, the combinatorial synthesis of drug libraries is common use, therefore efficient tools for the characterization of drug candidates and the extent of interaction between a drug and its target protein is a central question of analytical interest. While biological activity is tested today by enzyme assays, MS techniques attract more and more attention as an alternative for a rapid comparison of drug-target interactions. CE enables the separation of proteins and drug-enzyme complexes preserving their physiological activity in aqueous media. By hyphenating CE with ESI-MS/MS, the binding strength of enzyme inhibitors can be deduced from MS/MS experiments, which selectively release the inhibitor from the drug-target complex after CID. In this study, alpha-chymotrypsin (CT), a serine protease, was chosen as a model compound. Chymostatin is a naturally occurring peptide aldehyde binding to CT through a hemiacetal bond and electrostatic interaction. First, a CE separation was developed, which allows the analysis of alpha-CT and a chymotrypsin-chymostatin complex under MS-compatible conditions. The use of neutral-coated CE capillaries was mandatory to reduce analyte-wall interactions. ESI-quadrupole ion trap-MS was worked out to demonstrate the selective drug release after CID. Fragmentation of the drug-enzyme complex was monitored in dependence from the excitation energy in the ion trap, leading to the V(50) voltage that enables 50% complex fragmentation as a reference value for chymotrypsin-chymostatin complex. A stable CE-ESI-MS/MS setup was established, which preserves the drug-enzyme complexes during ionization-desolvation processes. With this optimized setup, different CT inhibitors could be investigated and compared.

Investigating metabolite–protein interactions: An overview of available techniques

“…Liquid chromatography (LC) [24] and gas chromatography (GC) [25] are commonly used. Recent advances in capillary electrophoresis (CE) [26] also make it an appealing choice complementing LC/GC.…”

“…Hoffmann et al used CE to separate chymotrypsin-chymostatin complexes from unbound chymotrypsin and impurities. They then selectively dissociated these enzyme-inhibitor complexes by using a collision-induced dissociation (CID) process in MS/MS, to analyze the binding strength of inhibitors [26]. CE has high efficiency and low sample consumption, but risks protein adsorption on the capillary.…”

Studies of metabolite–protein interactions: A review

“…Capillary electrophoresis (CE) is another separation method that can be used to examine metabolite-protein binding [9,37,40,41]. One way this method can be used is to separate the free and bound metabolites through the differences in their size-to-charge ratios.…”

“…One way this method can be used is to separate the free and bound metabolites through the differences in their size-to-charge ratios. This approach can be utilized alone to determine the affinity of metabolite-protein binding or combined with other methods such as mass spectrometry to examine this interaction [41]. One form of CE is affinity capillary electrophoresis (ACE), in which a biological molecule such as a protein is used as a running buffer additive, thus making it possible to obtain data on the interactions of solute components with this additive [40].…”

A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening by capillary electrophoresis

“…In fact, many new drugs developed between 1981 and 2002, particularly for treating cancer and infectious diseases originated from natural sources [12]. To study these complex sample matrices, screening assays based on liquid chromatography (LC), mass spectrometry (MS), and capillary electrophoresis (CE) were developed [13–15]. In most of these methods, an in-line enzyme bioreactor prepared by immobilizing the enzyme onto a solid support that was retained inside the LC or CE column was used.…”