Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A.
Botulinum neurotoxin serotype A (BoNT/A) is a proteolytic enzyme that induces muscle paralysis. It is a cause of food poisoning, a potential bioterrorist threat and, in low doses an emerging pharmaceutical product. No effective treatment is currently available for BoNT intoxication. Previously we developed a BoNT/A light chain enzyme assay using a peptide substrate based on the SNAP-25 protein target, with HPLC separation and UV detection of assay products, and applied the method to screen combinatorial peptide libraries for inhibitory activity to BoNT/A. We now report on development of a capillary electrophoresis laser-induced fluorescence (CE-LIF) method for measuring BoNT/A activity. The enzyme assay products were labeled with CBQCA dye followed by CE separation on a bare fused silica column in a HEPES-based buffer and LIF detection. All assay products were separated in CE within 8 min compared to incomplete separation of assay products within 1h by HPLC. The labeled products showed linear dependence of intensity versus concentration, and quantitative mole-fraction assignments. We used the CE-LIF method to screen combinatorial peptide libraries for potential modulating effects on BoNT/A peptidase activity. With some of the libraries, peptides co-migrated with assay products and interfered with quantitation. In such cases, interference was reduced by substituting sodium dodecyl sulfate (SDS) for Tween-20 in the running buffer. Separation in the capillaries then occurred by micellar electrokinetic chromatography (MEKC). The CE-LIF method is quick and lends itself to high-throughput or microfluidic formats.
Laing
Terrina Dickinson
TD
Canada West Biosciences Inc., 44 Inverness Dr. S.E., Calgary, Alta., Canada T2Z 3E4.
Marenco
Armando J
AJ
Moore
Diana M
DM
Moore
Graham J
GJ
Mah
David C W
DC
Lee
William E
WE
D000595:Amino Acid Sequence; D019274:Botulinum Toxins, Type A / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D020374:Chromatography, Micellar Electrokinetic Capillary; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D010446:Peptide Fragments / Q000302:isolation & purification; D019151:Peptide Library; D013050:Spectrometry, Fluorescence
D010446:Peptide Fragments; D019151:Peptide Library; D019274:Botulinum Toxins, Type A