On-Line Coupling of High-Performance Liquid Chromatography to a Continuous-Flow Enzyme Assay Based on Electrospray Ionization Mass Spectrometry

Abstract

Liquid chromatography (LC) was coupled on-line to a continuous-flow enzymatic assay using electrospray ionization mass spectrometry (ESI-MS) as readout for the screening of enzyme inhibitors in complex samples. Inhibitors were detected by changes in the concentration of the enzymatic reaction products, indicating the inhibition of enzymatic activity. The molecular masses of the inhibitors were determined with high certainty by using retention time matching and peak shape comparison. Due to the high matching accuracy, baseline separation of coeluting analytes was not necessary in order to identify the correct masses of the bioactive compounds. The continuous-flow system was successfully applied for the screening of complex samples, such as natural extracts. For a red clover extract, detection limits of 0.3-0.8 micromol/L were obtained. System validation was performed by determining the IC(50) values of four inhibitors in the flow-injection mode. The IC(50) values were in the 0.11-5.6 micromol/L range and correspond closely to data obtained by microtiter plate assays. Detection limits were in the range of 0.018-0.35 micromol/L in the flow-injection mode, and 0.075-0.75 micromol/L in the LC mode. These values are well below the typical compound concentrations (1-10 micromol/L) used in high-throughput screening. Together with an interday precision of 12.6%, these results demonstrate the applicability of the system for bioactivity screening of complex mixtures, generating both chemical and biological information on bioactive compounds in a single run.

MALDI-TOF MS as a label-free approach to rapid inhibitor screening

“…To date, nearly all the reports on the use of MS-based assays for compound screening rely on highly quantitative LC-MS approaches [14]. Some exceptions have included reports using porous siliconbased surfaces (DIOS) [15,16] and tailored target surfaces to enhance the interaction of ligands with target proteins (SAMDI) [17].…”

Real-time ESI-MS of Enzymatic Conversion: Impact of Organic Solvents and Multiplexing

“…As reviewed in Kool et al, advances in separation techniques and mass spectrometric detection technology lead to increasing importance of online coupled setups and have already been studied with acetylcholinesterase, phosphodiesterase, cytochrome P450, glutathione-S-transferase and cathepsin B. [24][25][26][27] An innovative tool for the effective coupling of chromatographic separation with enzymatic screening methods was described by Irth and coworkers who used high-temperature HPLC (HT-HPLC). 28 Compared to conventional HPLC, the amount of organic solvent needed for the separation can be decreased markedly and 10% organic phase is often sufficient for chromatographic separation.…”

Development of an at-line method for the identification of angiotensin-I inhibiting peptides in protein hydrolysates

“…All work aiming at the identification of ACE active ingredients published in literature so far is based on repeated off-line fractionation, activity measurement and identification [14][15][16]. A notable exception is the work by van Elswijk et al [8].…”