Antibodies for ubiquitinated proteins
The invention relates to particular ubiquitination epitopes, antibodies that specifically recognize and bind to ubiquitinated proteins and peptides (particularly after the ubiquitin is removed by proteolytic cleavage) and to methods of using these epitopes and antibodies.
1. A method of detecting a site of a diglycine (Gly-Gly) epitope linked to the epsilon amino group of a lysine in a protein present in a sample, wherein the method comprises
(i) cleaving protein(s) in the sample with a protease which cleaves at the C-terminus of Arg to form a mixture of cleavage peptides, wherein at least one cleavage peptide in the mixture comprises a lysine which is linked through its epsilon amino group to the C-terminus of a Gly-Gly dipeptide;
(ii) contacting the mixture of cleavage peptides with an antibody, wherein said antibody specifically binds to a Gly-Gly dipeptide linked to the epsilon amino group of a lysine, and said contacting is performed under conditions that allow said antibody to specifically bind to said at least one cleavage peptide; and
(iii) isolating said at least one cleavage peptide bound to said antibody from the mixture of cleavage peptides, thereby detecting a site of a diglycine (Gly-Gly) epitope linked to an epsilon amino group of a lysine in a protein present in said sample.
2. The method of claim 1 , wherein the diglycine (Gly-Gly) epitope linked to the epsilon amino group of a lysine comprises the following structure:
wherein: X 1 is hydrogen or a first peptide sequence;
X 2 is hydroxy or a second peptide sequence; and
Y is a peptide having Arg at its C-terminus, wherein the Y peptide is not a full-length protein.
3. The method of claim 2 , wherein Y is Leu-Arg or STLHLVLRLR (SEQ ID NO:344).
4. The method of claim 1 , wherein the protease cleaves ubiquitin and also cleaves protein(s) in the sample.
5. The method of claim 1 , wherein the amino acid sequence of said peptide is determined.
6. The method of claim 1 , wherein the protease is trypsin or Arg-C.
7. The method of claim 5 , wherein sequencing of said peptide is performed by a method comprising mass spectrometry.
8. The method of claim 1 , further comprising identifying the diglycine modified lysine site in the cleavage peptide.
9. The method of claim 1 , further comprising identifying the protein from which the cleavage peptide was derived.
10. The method of claim 9 , further comprising isolating the protein from which the cleavage peptide was derived.
11. The method of claim 1 , wherein step (i) is performed in the presence of H 2 18 O.
12. The method of claim 11 , wherein the amount or proportion of cleavage peptides labeled with 18 O is determined.
13. The method of claim 1 , wherein the sample is a protein sample, body fluid, tissue sample, cell lysate, fractionated cellular material, cellular extract, cell culture supernatant, or cultured cells.
14. The method of claim 13 , wherein the body fluid is saliva, mucous, sweat, whole blood, serum, urine, amniotic fluid, genital fluid, fecal material, marrow, plasma, spinal fluid, pericardial fluid, gastric fluid, abdominal fluid, peritoneal fluid, pleural fluid, synovial fluid, cyst fluid, cerebrospinal fluid, lung lavage fluid, lymphatic fluid, tears, prostatitc fluid, tissue extract, or glandular secretion.
15. The method of claim 1 , wherein the sample is obtained from a mammal fed a diet containing an isotopically-labeled amino acid.
16. The method of claim 1 , wherein the sample is obtained from culture media containing an isotopically-labeled amino acid.
17. The method of claim 1 , wherein the sample comprises a selected cell or tissue type and the method further comprises identifying substantially all proteins that contain the epitope in the cell or tissue type to yield a profile of proteins from the cell or tissue type that contain the epitope.
18. The method of claim 17 , further comprising comparing the profile of proteins from the cell or tissue type that contain the epitope with a profile of proteins that contain the epitope in the cell or tissue type after treatment or exposure of the selected cells or tissues to a drug or test agent.
19. The method of claim 17 , further comprising comparing the profile of proteins from the cell or tissue type that contain the epitope with a profile of proteins that contain the epitope from cells with a mutation in, or an amplification of, a gene encoding a ubiquitin ligase, a ubiquitin conjugating enzyme or a ubiquitin activating enzyme.
20. The method of claim 19 , wherein the mutation substantially eliminates expression or function of an E3 ubiquitin ligase, an E2 ubiquitin conjugating enzyme or an E1 ubiquitin activating enzymes.
21. The method of claim 1 , wherein the diglycine (Gly-Gly) epitope site is a ubiquitinated site.