C-terminal sequencing of peptides which may include proline

Details C-terminal sequencing of peptides which may include proline C-terminal sequencing of peptides which may include proline

1995-11-09

1999-12-07

Alexander, Lyle A.

Chemistry: analytical and immunological testing

Peptide, protein or amino acid

Amino acid or sequencing procedure

530345, 530402, G01N 3368

Patent

active

059982136

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to a method for the degradation from the C-terminus of peptides which may include a proline residue.


BACKGROUND OF THE INVENTION

Kenner, et al. J. Chem. Soc. 673-678 (1953) describes a C-terminal degradation experiment which required 110 hours to quantitatively form a thiohydantoin amino acid. Bailey U.S. Pat. No. 5,180,807 describes an improvement in which diphenyl phosphoroisothiocyanatidate is used concurrently with a heterocyclic amine, such as pyridine.
Application Ser. No. 08/094,024 illustrates sequential use of diphenyl phosphoroisothiocyanatidate and a heterocyclic amine for C-terminal peptide degradation. In a first step, the peptide which is preferably bound to a solid phase is converted to a carboxylate salt by triethylamine or similar base. In a second step, the carboxylate is reacted with diphenyl phosphoroisothiocyanatidate. In a third step, a heterocyclic amine such as pyridine is added.


SUMMARY OF THE INVENTION

This invention provides a method for the C-terminal degradation of peptides which may include a proline residue. The method of the invention entails (1) formation of a carboxylate on the C-terminal amino acid of the peptide to be sequenced, (2) reaction of the carboxylated peptide with diphenyl phosphoroisothiocyanatidate and a heterocyclic amine to produce a thiohydantoin derivative, (3) protonating the thiohydantoin derivative, and (4) cleaving the protonated thiohydantoin derivative to produce a shortened peptide and a thiohydantoin derivative of the C-terminal amino acid of the peptide to be sequenced.
The polypeptides to be sequenced are preferably either non-covalently applied to the porous tetrafluoroethylene (Zitex) or covalently attached to carboxylated polyethylene (PE--COOH). See application Ser. No. 07/576,943 and U.S. Pat. No. 5,180,807.


DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of one C-terminal sequencer useful in the practice of the invention.
FIG. 2 illustrates the practice of the invention to sequence YGGFL covalently coupled to carboxylic acid modified polyethylene (PE--COOH). R4 is gas phase pyridine.
FIGS. 3A-3C illustrates the practice of the invention to sequence YGGFL covalently coupled to PE--COOH. R4 is a solution of tetrazole in dimethylformamide.
FIGS. 4A to 4F illustrate the practice of the invention to sequence YGGFL covalently coupled to PE--COOH. R4 is a solution of tetrazole in acetonitrile.
FIG. 5 illustrates the practice of the invention to sequence LAP covalently coupled to PE--COOH.
FIG. 6 illustrates the practice of the invention to sequence AGSE covalently coupled to PE--COOH.
FIG. 7 illustrates the practice of the invention to sequence Superoxide Dismutase non-covalently coupled to polytetrafluoroethylene (Zitex).
FIG. 8 illustrates the practice of the invention to sequence Ribonuclease A non-covalently coupled to polytetrafluoroethylene (Zitex).
FIG. 9 illustrates the practice of the invention to sequence hemoglobin .alpha. chain non-covalently coupled to polytetrafluoroethylene (Zitex).


DETAILED DESCRIPTION OF THE INVENTION

The invention provides a preferably sequential four-step method for degradation of a peptide which may include proline.


Step 1. Carboxylation of the Peptide

A carboxylate is formed at the C-terminus of the peptide to be sequenced by reaction with an organic or inorganic base. The specific base utilized is not critical. The carboxylation is preferably carried out by reaction of the peptide to be sequenced with a solution of the selected base in an appropriate solvent. Tertiary trialkyl amines are preferred. Primary and secondary alkyl amines may also be utilized. Alkali metal bases such as sodium or potassium hydroxide are effective and may be utilized in an aqueous solution. Sodium trimethylsilanolate in methyl alcohol solution is appropriate.
When utilized in aqueous solution, alkyl amines such as triethylamine are preferably present in a concentration of about 5% by volume. When such amines are utilized in an organic solvent solution, a concentration o

REFERENCES:
patent: 5254475 (1993-10-01), Bailey
patent: 5432092 (1995-07-01), Bailey et al.
Bailey et al., Protein Science:1(12), pp. 1622-1633 (1992).
Boyd et al., Analytical Biochemistry, 206, pp. 344-352 (1992).

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