Screening of combinatorial libraries for substrate preference by mass spectrometry.
We present a rapid screening method for monitoring enzyme specificity using both combinatorial chemistry and mass spectrometry where, as an example, the substrate specificity of peptidylglycine alpha-amidating enzyme was determined and compared against a conventional quantitative technique. Whereas alternative methods for library screening are generally limited to certain enzymes and can present difficulties in the synthesis or derivatization of potential substrates, the approach we call chirality-based isotope labeling for a library of substrates (CHILLS) does not fall short to such limitations, since we exploit the inherent stereospecificity of enzymes to determine preferred substrates. Additionally, the CHILLS method generates accurate results, as compared to typical screening procedures that require tedious method development, because the synthesized library contains a structurally similar internal standard for each individual library component in order to quantitate the progress of enzymatic reactions.
Stevens
Stanley M
SM
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida 32610-0485, USA.
Prokai-Tatrai
Katalin
K
Prokai
Laszlo
L
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D020650:Combinatorial Chemistry Techniques; D004798:Enzymes / Q000378:metabolism*; D007536:Isomerism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D019151:Peptide Library; D013379:Substrate Specificity
D004798:Enzymes; D009097:Multienzyme Complexes; D019151:Peptide Library; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase