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Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Nov 07; 843(2):240-6.JC

Abstract

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.

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Publisher Full Text

Authors+Show Affiliations

Laing TD
Canada West Biosciences Inc., 44 Inverness Dr. S.E., Calgary, Alta., Canada T2Z 3E4.
Marenco AJ
No affiliation info available
Moore DM
No affiliation info available
Moore GJ
No affiliation info available
Mah DC
No affiliation info available
Lee WE
No affiliation info available

MeSH

Amino Acid SequenceBotulinum Toxins, Type AChromatography, High Pressure LiquidChromatography, Micellar Electrokinetic CapillaryElectrophoresis, CapillaryLasersPeptide FragmentsPeptide LibrarySpectrometry, Fluorescence

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

16828348

Citation

Laing, Terrina Dickinson, et al. "Capillary Electrophoresis Laser-induced Fluorescence for Screening Combinatorial Peptide Libraries in Assays of Botulinum Neurotoxin A." Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 843, no. 2, 2006, pp. 240-6.
Laing TD, Marenco AJ, Moore DM, et al. Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;843(2):240-6.
Laing, T. D., Marenco, A. J., Moore, D. M., Moore, G. J., Mah, D. C., & Lee, W. E. (2006). Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 843(2), 240-6.
Laing TD, et al. Capillary Electrophoresis Laser-induced Fluorescence for Screening Combinatorial Peptide Libraries in Assays of Botulinum Neurotoxin A. J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Nov 7;843(2):240-6. PubMed PMID: 16828348.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A. AU - Laing,Terrina Dickinson, AU - Marenco,Armando J, AU - Moore,Diana M, AU - Moore,Graham J, AU - Mah,David C W, AU - Lee,William E, Y1 - 2006/07/07/ PY - 2005/10/20/received PY - 2006/06/06/revised PY - 2006/06/07/accepted PY - 2006/7/11/pubmed PY - 2006/12/30/medline PY - 2006/7/11/entrez SP - 240 EP - 6 JF - Journal of chromatography. B, Analytical technologies in the biomedical and life sciences JO - J Chromatogr B Analyt Technol Biomed Life Sci VL - 843 IS - 2 N2 - 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. SN - 1570-0232 UR - https://www.unboundmedicine.com/medline/citation/16828348/Capillary_electrophoresis_laser_induced_fluorescence_for_screening_combinatorial_peptide_libraries_in_assays_of_botulinum_neurotoxin_A_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1570-0232(06)00471-5 DB - PRIME DP - Unbound Medicine ER -