Analysis of Ubiquitinated Polypeptides

• US 20200131254A1
• Filed: 07/11/2019
• Published: 04/30/2020
• Est. Priority Date: 07/11/2006
• Status: Active Grant

• Current Assignee: Cell Signaling Technology Incorporated
• Original Assignee: Cell Signaling Technology Incorporated
• Inventors: Comb, Michael J., Rush, II, John Edward, Li, Jing, Guo, Ailan
• Granted Patent: US 11,352,418 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The disclosure relates to antibody reagents that specifically bind to peptides carrying a ubiquitin remnant from a digested or chemically treated biological sample. The reagents allow the technician to identify ubiquitinated polypeptides as well as the sites of ubiquitination on them. The reagents are preferably employed in proteomic analysis using mass spectrometry. The antibody reagents specifically bind to the remnant of ubiquitin (i.e., a diglycine modified epsilon amine of lysine) left on a peptide which as been generated by digesting or chemically treating ubiquitinated proteins. The inventive antibody reagents'"'"' affinity to the ubiquitin remnant does not depend on the remaining amino acid sequences flanking the modified (i.e., ubiquitinated) lysine, i.e., they are context independent.

Claims

1. An isolated binding partner that specifically binds a ubiquitin remnant peptide but does not specifically bind a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant.

2. An isolated antibody that specifically binds a ubiquitin remnant peptide but does not specifically bind a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant.

3. The antibody of claim 2, wherein the antibody is a monoclonal antibody.

4. The antibody of claim 2, wherein the antibody is a polyclonal antibody.

5. The antibody of claim 2, wherein the antibody is a human antibody.

6. The antibody of claim 2, wherein the antibody is a chimeric antibody.

7. The antibody of claim 2, wherein the antibody has a dissociation constant (kD) of 10⁻

7 M or less for the ubiquitin remnant peptide.

8. The antibody of claim 2, wherein the antibody has a dissociation constant (kD) of 10⁻

9 M or less for the ubiquitin remnant peptide.

9. The antibody of claim 2, wherein the antibody has an off rate (koff) of 10⁻

3/sec or less.

10. The antibody of claim 2, wherein the antibody has an off rate (koff) of 10⁻

4/sec or less.

12. The antibody of claim 2, wherein the antibody specifically binds a ubiquitinated lysine residue in the ubiquitin remnant peptide regardless of the peptide sequence surrounding the ubiquitinated lysine residue.

13. The antibody of claim 2, wherein the antibody is a monoclonal antibody.

14. The antibody of claim 2, wherein the ubiquitin remnant is a diglycine modified epsilon amine of lysine.

15. The antibody of claim 2, wherein the ubiquitin remnant is a portion of a ubiquitinated polypeptide that remains attached to a digestion product of the ubiquitinated polypeptide that has been exposed to trypsin.

11. (canceled)

16. (canceled)

17. (canceled)

18. A method of producing a purified motif-specific, context-independent antibody that specifically binds a motif consisting of a ubiquitin remnant peptide but does not specifically bind a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacks the ubiquitin remnant, wherein the ubiquitin remnant is a diglycine modified epsilon amine of lysine, wherein the method comprises (a) constructing a degenerate peptide library comprising (i) the motif and (ii) a plurality of degenerate amino acids flanking said motif;

(b) immunizing a host with said peptide library to raise a context-independent antibody specific for all or part of said motif; and

(c) isolating antisera from said host, and purifying the motif-specific, context-independent antibody of step (b) from said antisera.

19. The method of claim 18, wherein the antibody is a monoclonal antibody.

20. The method of claim 18, wherein said peptide library is from 6 to 20 amino acids long.

21. The method of claim 18, wherein said peptide library is from 6 to 14 amino acids long.