Method for protein and peptide sequencing using derivatized glass supports

• US 4,992,383 A
• Filed: 08/05/1988
• Issued: 02/12/1991
• Est. Priority Date: 08/05/1988
• Status: Expired due to Fees

• Current Assignee: Beckman Instruments, Inc. (Danaher Corporation)
• Original Assignee: Porton Instruments Incorporated
• Inventors: Farnsworth, Vincent
• Primary Examiner(s): CANNON, JAMES
• Assistant Examiner(s): Vassilatos, Thalia P.
• Application Number:
  US07/228,524
• Time in Patent Office: 921 Days
• Field of Search: 436/89, 436/178, 435/6
• US Class Current: 436/89
• CPC Class Codes: C07K 1/128 sequencing
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

Peptides or proteins are sequenced by stepwise degradation while immobilized on a glass support derivatized with a silica-binding substance bearing a free acid group, especially a sulfonic acid group. The support is preferably derivatized with 2-(4-chlorosulfonyl phenyl) ethyl trimethoxysilane.

Peptide sequencing performance is improved if the support is also derivatized with a monomeric silica-binding substance bearing a free quaternary ammonium group, such as N-trimethyoxysilyl propyl -N,N,N-trimethyl ammonium chloride.

Claims

1. In a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the improvement which comprises providing a glass support derivatized with a silica-binding substance having a free acid group, said acid having a pKa<

1, said derivatized support being capable of retaining said peptide or protein.

2. The method of claim 1 wherein said acid group is selected from the group consisting of phosphoric, picric, hydrochloric, trifluoroacetic, trichloracetic, chromic, hydroiodic, pyrophosphoric, sulfonic and halosulfonic acids.

3. The method of claim 1 in which the acid group is a sulfonic or halosulfonic acid.

4. The method of claim 3 in which the support is prepared by incubating a glass support with a 4-chlorosulfonyl phenyl alkyl alkoxysilane.

5. The method of claim 4 in which the silane is 2-(4-chlorosulfonyl phenyl) ethyl trimethyoxysilane.

6. The method of claim 4 in which the silane is 3-(trimethylsilyl)-1-propane sulfonic acid.

10. The method of claim 1 wherein the silica-binding substance is an organosilane having the formula R_n SiX.sub.(4-n), where n is 1 to 3, X is a hydrolyzable group suitable for covalently binding the substance to the glass support, and R is a nonhydrolyzable organic radical bearing said free acid group, said derivatized support being capable of retaining the peptide or protein.

11. The method of claim 10 wherein X is selected from the group consisting of alkoxy, acyloxy, amine and chlorine.

7. In a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the improvement which comprises immobilizing the peptide or protein on a glass support derivatized with a first silica-binding substance having a free sulfonic or halosulfonic acid group, and a second silica-binding substance having a free quaternary ammonium group, wherein said support is capable of retaining a peptide or protein.

8. The method of claim 7 in which the first substance is 2-(4-chlorosulfonyl phenyl)ethyl trimethoxysilane, and the second substance is N-trimethoxysilyl propyl-N,N,N-trimethyl ammonium chloride.

9. The method of claim 8 in which the substance is N-trimethoxysilyl propyl-N,N,N-trimethyl ammonium chloride.

12. In a method of sequencing a peptide or protein which comprises immobilizing the peptide or protein on a support and sequencing the peptide or protein by stepwise degradation, the improvement which comprises providing a glass support derivatized with an organosilane having the formula R_n SiX.sub.(4-n), where n is 1 to 3, X is a hydrolyzable group suitable for covalently bonding the substance to the glass support, and R is a nonhydrolyzable organic radical bearing a quaternary ammonium group, said derivatized support being capable of retaining the peptide or protein.

13. The method of claim 12 wherein X is selected from the group consisting of alkoxy, acyloxy, amine and chlorine.