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Inhibitor Screening Using Immobilized Enzyme Reactor Chromatography/Mass Spectrometry
journal contribution
posted on 2005-12-01, 00:00 authored by Richard J. Hodgson, Travis R. Besanger, Michael A. Brook, John D. BrennanWe describe the coupling of capillary-scale monolithic
enzyme reactor columns directly to a tandem mass
spectrometer for screening of enzyme inhibitors. A two-channel nanoLC system is used to continuously infuse
substrate or substrate/inhibitor mixtures through the
column, allowing continuous variation of inhibitor concentration by simply altering the ratio of flow from the two
pumps. In the absence of inhibitor, infusion of substrate
leads to formation of product, and both substrate and
product ions can be simultaneously monitored in a
quantitative manner by MS/MS. The presence of inhibitor
leads to a decrease in product and an increase in
substrate concentration in the column eluent. Knowing
the product/substrate ratio and the total analyte concentration (P + S), the concentration of product eluting, and
hence the relative enzyme activity, can be determined.
Both IC50 and KI values can then be obtained by direct
MS detection of the effect of inhibitors on relative activity.
Inhibitor screening is demonstrated using reusable, sol−gel derived, monolithic capillary columns containing adenosine deaminase, directly interfaced to ESI-MS/MS.
On-column enzyme activity was assessed by monitoring
inosine and adenosine elution. It is shown that the method
can be used for automated screening of the effects of
compound mixtures on ADA activity and to determine the
KI value of the known inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, even when the compound is present within
a mixture.
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Keywords
enzyme activityADA activitycompound mixturesInhibitor screeningmonitoring inosineadenosine elutioncapillary columnstandem mass spectrometercolumn eluentInhibitor Screeningproduct ionsIC 50adenosine deaminasesubstrate concentrationanalyte concentrationMS detectionproduct elutingenzyme reactor columnsenzyme inhibitorsinhibitor concentration
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