Analysis of ubiquitinated polypeptides

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.

1 . A method for determining the presence of at least one ubiquitinated polypeptide in a biological sample comprising:
 a. Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample;   b. Contacting the hydrolyzed sample with a substrate comprising at least one immobilized binding partner; wherein the at least one immobilized binding partner 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;   c. Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner;   d. Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution;   e. Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is derived from saliva, mucous, tears, blood, serum, lymph fluids, buccal cells, mucosal cells, biopsy tissue, cerebrospinal fluid, semen, feces, plasma, urine, a suspension of cells, or a suspension of cells and viruses. 
     
     
         3 . The method of  claim 1 , wherein the hydrolyzing agent is an enzyme selected from the group consisting of trypsin, Lysine-C endopeptidase (LysC), arginine-C endopeptidase (ArgC), Asp-N, glutamic acid endopeptidase (GluC) and chymotrypsin; or combinations thereof 
     
     
         4 . The method of  claim 1 , wherein the substrate comprises a gel matrix. 
     
     
         5 . The method of  claim 1 , wherein the substrate comprises polymer beads. 
     
     
         6 . The method of  claim 1 , wherein the at least one immobilized binding partner comprises an antibody or ubiquitin remnant peptide binding fragment thereof. 
     
     
         7 . The method of  claim 6 , wherein the antibody is a polyclonal antibody. 
     
     
         8 . The method of  claim 6 , wherein the antibody is a monoclonal antibody. 
     
     
         9 . The method of  claim 1  wherein a portion of the elution solution is analyzed by mass spectrometry. 
     
     
         10 . The method of  claim 1 , wherein step (e) is performed by tandem MS. 
     
     
         11 . The method of  claim 1 , wherein the amino acid sequence of at least one ubiquitin remnant peptide present in the elution solution, is determined. 
     
     
         12 . The method of  claim 11 , wherein the sequence is compared to the sequence of the ubiquitinated polypeptide to determine the site of ubiquitination. 
     
     
         13 . The method of  claim 1 , wherein the elution solution further comprises at least one standard peptide, wherein the at least one standard peptide has the substantially the same amino acid sequence as the at least one distinct peptide but a different measured accurate mass. 
     
     
         14 . The method of  claim 1  wherein ubiquitinated polypeptide comprises a ubiquitin-like protein. 
     
     
         15 . The method of  claim 1  the ubiquitin-like protein is selected from the group consisting of NEDD8 and ISG15. 
     
     
         16 . The method of  claim 1 , wherein the hydrolyzing agent is trypsin. 
     
     
         17 . The method of  claim 1 , wherein the biological sample is depleted of a protein selected from the group consisting of albumin, IgG, IgA, transferrin, haptoglobin, and anti-trypsin; or combinations thereof, prior to step (a). 
     
     
         18 . 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. 
     
     
         19 . A method for determining whether a patient is has or is likely to have or develop a disease associated with a least one ubiquitinated polypeptide comprising:
 a. Obtaining a biological sample from the patient;   b. Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving the ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample;   c. Contacting the hydrolyzed sample with a substrate comprising at least one immobilized binding partner; wherein the at least one immobilized binding partner 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;   d. Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner;   e. Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; and   f. Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide;   g. Determining that the patient is has or is likely to have or develop a disease associated with a least one ubiquitinated polypeptide if the least one ubiquitinated polypeptide is present in the biological sample.   
     
     
         20 . A method for determining whether a disease is associated with at least one ubiquitinated polypeptide comprising:
 a. Obtaining a biological sample from a patient having the disease;   b. Contacting the sample with at least one hydrolyzing agent, wherein the hydrolyzing agent is capable of cleaving a ubiquitinated polypeptide to produce at least one ubiquitin remnant peptide, to obtain a hydrolyzed sample;   c. Contacting the hydrolyzed sample with a substrate comprising an at least one immobilized binding partner; wherein the at least one immobilized binding partner 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;   d. Removing the hydrolyzed sample from the substrate in a manner such that the at least one ubiquitin remnant peptide would remain bound to the immobilized binding partner;   e. Contacting the substrate with an elution solution, where in the least one ubiquitin remnant peptide would dissociate from the immobilized binding partner into the elution solution; and   f. Determining the presence of a least one ubiquitinated polypeptide in the biological sample when the elution solution contains the at least one least ubiquitin remnant peptide;   g. Determining that the disease is associated with the presence of the at least one ubiquitinated polypeptide if the least one ubiquitinated polypeptide is absent in the biological sample of a healthy individual.