The sequential analysis of peptides and proteins from the amino-terminus has been well established. On the contrary, a satisfactory sequential analysis of peptides and proteins from the carboxy-terminus has not been demonstrated. Such a procedure, in addition to complementing amino- terminal degradation, would allow the direct analysis of polypeptides and proteins with blocked amino-termini and would be invaluable for the detection of post-translational processing at the carboxy-terminus of expressed gene products from known DNA sequences. Of the chemical methods proposed, the method based on the derivatization of the carboxy-terminal amino acid to a thiohydantoin with thiocyanate based reagents has been the most widely studied and appears to be the most attractive due to its similarity to the current methods of N-terminal sequencing. Preliminary work in our laboratory has examined in detail the generality of this method for the sequential degradation of the twenty naturally occurring amino acids in the solution phase. Each cycle of a degradation consists of three steps: activation of the carboxy-terminal carboxylic group, derivatization to a thiohydantoin amino acid, and specific hydrolysis of the derivatized C-terminal amino acid to a thiohydantoin amino acid derivative and a shortened polypeptide or protein capable of continued degradation. Current limitations of this method include: a limited number of cycles due to sample losses inherent during manual manipulation, a reduced yield when aspartic acid is encountered during the degradation, and the inability of the method to degrade proline when it is at the C-terminus of peptides or proteins. Application of this chemistry to the solid phase and to automation has the potential to permit extended sequential degradation from the C-terminus of peptides and proteins with minimal sample loss. Advantages to the solid phase approach include the ability to use reagents and solvents optimal for sequencing without causing sample washout and the capability to efficiently wash the sample to remove reaction byproducts which might otherwise interfere with identification of the released thiohydantoin amino acids. Preliminary work in our laboratory directed toward the application of C-terminal sequencing to the solid phase and toward automation has resulted in some success and has outlined some of the challenges ahead. The ultimate goal of this research is the development of chemistry that will allow the sequential degradation of proteins and peptides, composed of any of the twenty naturally occurring amino acids, from the C-terminus with the same speed, sensitivity, and number of repetitive cycles currently possible with existing N-terminal methods.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046022-03
Application #
2183611
Study Section
Biochemistry Study Section (BIO)
Project Start
1992-02-01
Project End
1996-01-31
Budget Start
1994-02-01
Budget End
1996-01-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010