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Double coupling Edman chemistry for high-sensitivity automated protein sequencing
I D Ireland1, D F Lewis, X F Li
1Department of Chemistry, University of Alberta, Edmonton, Canada.
Journal of Protein Chemistry
|July 1, 1997
Summary
Capillary electrophoresis with laser-induced fluorescence detection offers highly sensitive protein sequencing analysis. A new miniaturized sequencer is developed for compatibility with this advanced detection method.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biotechnology
Background:
- Protein sequencing is crucial for understanding protein function.
- Traditional methods face limitations in sensitivity and sample volume.
- Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) shows potential for high-sensitivity analysis.
Purpose of the Study:
- To develop a CE-LIF method for analyzing protein sequencing products.
- To achieve ultra-low limits of detection for fluorescein thiohydantoin (FTH) amino acids.
- To develop a compatible miniaturized sequencer for integrated protein analysis.
Main Methods:
- Development of a capillary electrophoresis separation for (FTH)-amino acid derivatives.
- Utilizing laser-induced fluorescence detection for sensitive analyte quantification.
- Engineering a miniaturized automated sequencer for on-line coupling with CE-LIF.
Main Results:
- Achieved 10 zmol (3 sigma) limits of detection for FTH amino acids using CE-LIF.
- Successfully separated FTH-amino acid products from 18 of 20 coded amino acids.
- Demonstrated the feasibility of a miniaturized sequencer compatible with CE-LIF.
Conclusions:
- CE-LIF is a powerful technique for sensitive protein sequencing product analysis.
- The developed miniaturized sequencer addresses volume limitations of commercial instruments.
- Integration of the miniaturized sequencer and CE-LIF enables highly sensitive, automated protein sequencing.