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N-terminal sequence analysis of polypeptide at the picomole level
The Biochemical Journal
|December 1, 1981
Summary
This study presents a manual N-terminal sequencing method for polypeptides, achieving subnanomole sensitivity. The technique uses a double-coupling approach and colored derivatives for sensitive amino acid identification, enabling peptide and protein sequencing without automated equipment.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Accurate N-terminal sequencing is crucial for protein identification and functional studies.
- Existing methods often require larger sample amounts or specialized automated equipment.
Purpose of the Study:
- To develop a sensitive, manual method for N-terminal polypeptide sequencing.
- To enable analysis of small peptide and protein quantities without automated sequenators or radioactive labels.
Main Methods:
- Stepwise degradation of polypeptides using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method.
- Identification of released amino acid derivatives (dimethylaminoazobenzenethiohydantoins) via reversed-phase high-pressure liquid chromatography (RP-HPLC).
- Development of a comprehensive RP-HPLC method for analyzing all amino acid derivatives and by-products.
Main Results:
- Achieved subnanomole sensitivity for N-terminal sequence analysis.
- Demonstrated detection of colored amino acid derivatives at 1 pmol sensitivity using visible light detection.
- Successfully analyzed complete peptide sequences and N-terminal protein sequences with less than 1 nmol of material.
Conclusions:
- The described manual method provides a sensitive and accessible alternative for polypeptide N-terminal sequencing.
- This technique is valuable for researchers with limited sample amounts or access to advanced sequencing instrumentation.
- The method allows for the complete sequencing of peptides and N-terminal sequencing of proteins efficiently.