Analysis of ubiquitinated polypeptides

• US 9,181,326 B2
• Filed: 04/04/2013
• Issued: 11/10/2015
• Est. Priority Date: 06/21/2001
• Status: Active Grant

• Current Assignee: Cell Signaling Technology Incorporated
• Original Assignee: Cell Signaling Technology Incorporated
• Inventors: Rush, John Edward II, Li, Jing, Guo, Ailan
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US13/856,933
• Publication Number:
  US 20130245237A1
• Time in Patent Office: 950 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: G01N 2440/36 addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Abstract

The invention 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 antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant, wherein the antibody comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, and SEQ ID NO;

8.

2. The isolated antibody of claim 1, wherein the antibody comprises a heavy chain sequence comprising SEQ ID NO:

1.

3. The isolated antibody of claim 1, wherein the antibody comprises a light chain sequence comprising SEQ ID NO:

2.

6. The isolated antibody of claim 2, wherein the antibody comprises a light chain sequence comprising SEQ ID NO:

2.

4. An isolated antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant, wherein the antibody comprises the variable region of the heavy chain set forth in SEQ ID NO:

1.

7. The isolated antibody of claim 4, wherein the antibody comprises the variable region of the light chain set forth in SEQ ID NO:

2.

5. An isolated antibody that preferentially binds a ubiquitin remnant peptide over a peptide having the same amino acid sequence as the ubiquitin remnant peptide but lacking a ubiquitin remnant, wherein the antibody comprises the variable region of the light chain set forth in SEQ ID NO:

2.