LABELING AND DETECTION OF POST TRANSLATIONALLY MODIFIED PROTEINS

• US 20070249014A1
• Filed: 02/12/2007
• Published: 10/25/2007
• Est. Priority Date: 02/10/2006
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: AGNEW, Brian, GEE, Kyle, NYBERG, Tamara
• Application Number:
  US11/674,140
• Publication Number:
  US 20070249014A1
• Time in Patent Office: Days
• US Class Current: 435/68.100
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 14/47 from mammals
• 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: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 302/01096 Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label
• CPC Class Codes: Y10T 436/17 Nitrogen containing

Abstract

Provided in certain embodiments are new methods for forming azido modified biomolecule conjugates of reporter molecules, carrier molecules or solid support. In other embodiments are provided methods for enzymatically labeling a biomolecules with an azide group.

Claims

1. A method of forming glycoprotein conjugate, wherein the method comprises:

a) contacting the glycoprotein with UDP-GalNAz in the presence of a Gal T enzyme to form an azido modified glycoprotein;

b) contacting the azido modified glycoprotein with a reporter molecule, carrier molecule or solid support that comprises an azide reactive moiety to form glycoprotein reporter molecule, carrier molecule or solid support conjugate.

2. The method according to claim 1, wherein the azide reactive moiety is a terminal alkyne or a terminal triaryl phosphine.

3. The method according to claim 1, wherein the reporter molecule is a xanthene, cyanine, coumarin, borapolyazaindacene or pyrene dye.

4. The method according to claim 1, wherein the reporter molecule is an enzyme substrate or hapten.

5. The claim according to claim 1, wherein the azido modified glycoprotein is an antibody.

6. The method according to claim 1, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

7. The method according to claim 1, wherein the carrier molecule comprises an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

8. The method according to claim 1, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

9. The method according to claim 1, wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

10. A method of forming a glycoprotein conjugate, wherein the method comprises:

a) contacting a protein with an azido modified carbohydrate in the presence of an enzyme that will transfer the azido modified carbohydrate to the protein to form an azido modified glycoprotein;

b) contacting the azido modified glycoprotein with a reporter molecule, carrier molecule or solid support that comprises a terminal alkyne to form glycoprotein reporter molecule, carrier molecule or solid support conjugate.

11. A method of forming a glycoprotein conjugate, wherein the method comprises:

a) contacting a protein with a terminal alkyne modified carbohydrate in the presence of an enzyme that will transfer the terminal alkyne modified carbohydrate to the protein to form a terminal alkyne modified glycoprotein;

b) contacting the terminal alkyne modified glycoprotein with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form glycoprotein reporter molecule, carrier molecule or solid support conjugate.

12. A method for forming a phosphoprotein conjugate, wherein the method comprises:

a) contacting a protein with a terminal alkyne modified phosphate in the presence of an enzyme that will transfer the terminal alkyne modified phosphate to the protein to form a terminal alkyne modified phosphoprotein;

b) contacting the terminal alkyne modified phosphoprotein with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form phosphoprotein reporter molecule, carrier molecule or solid support conjugate.

13. A method for forming a phosphoprotein conjugate, wherein the method comprises:

a) contacting a protein with an azido modified phosphate in the presence of an enzyme that will transfer the azido modified phosphate to the protein to form a azido modified phosphoprotein;

b) contacting the azido modified phosphoprotein with a reporter molecule, carrier molecule or solid support that comprises a terminal alkyne to form phosphoprotein reporter molecule, carrier molecule or solid support conjugate.

14. A method for forming a phosphoprotein conjugate, wherein the method comprises:

a) contacting a phosphoprotein with a base solution that removes phosphates from threonine and serine residues of the phosphorylated protein to form a protein comprising dehydroalanine or dehydroamino-2-butyric acid;

b) contacting the dehydroalanine or dehydroamino-2-butyric acid with a compound that comprises a thiol or amine group and an azide or terminal alkyne to form an azido or alkyne modified protein;

c) contacting the terminal alkyne or azido modified phosphoprotein with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form phosphoprotein reporter molecule, carrier molecule or solid support conjugate.

15. A method for forming a post translationally modified protein conjugate, wherein the method comprises:

a) contacting a protein with an azido modified post translational moiety in the presence of an enzyme that will transfer the azido modified post translational moiety to the protein to form a azido modified protein;

b) contacting the azido modified protein with a reporter molecule, carrier molecule or solid support that comprises a terminal alkyne to form phosphoprotein reporter molecule, carrier molecule or solid support conjugate.

16. A method for forming a post translationally modified protein conjugate, wherein the method comprises:

a) contacting a protein with a terminal alkyne modified post translational moiety in the presence of an enzyme that will transfer the terminal alkyne modified post translational moiety to the protein to form a terminal alkyne modified protein;

b) contacting the terminal alkyne modified protein with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form phosphoprotein reporter molecule, carrier molecule or solid support conjugate.

17. A method of detecting an azido modified biomoelcule, comprising:

a) forming an azide-alkyne cycloaddition reaction mixture comprising;

a reporter molecule that comprises a terminal alkyne moiety;

an azido modified biomoelcule;

copper ions;

at least one reducing agent; and

a copper chelator;

b) incubating the azide-alkyne cycloaddition reaction mixture for a sufficient amount of time to form a biomoelcule-reporter molecule conjugate;

c) separating the biomoelcule-reporter molecule conjugate by size and/or weight of the glycoprotein-reporter molecule conjugate to form a separated biomoelcule-reporter molecule conjugate;

d) illuminating the separated biomoelcule-reporter molecule conjugate with an appropriate wavelength to form an illuminated biomoelcule-reporter molecule conjugate;

e) observing the illuminated biomolecule-reporter molecule conjugate wherein the glycoprotein is detected.

18. The method according to claim 17, wherein the biomolecule is carbohydrate, a nucleic acid, a protein, or a peptide.

19. The method according to claim 17, wherein the biomolecule is a glycoprotein.

20. The method according to claim 17, wherein the reporter molecule is xanthene, cyanine, coumarin, borapolyazaindacene or pyrene dye.

21. The method according to claim 17, wherein the reporter molecule is an enzyme substrate, fluorescent protein or hapten.

22. The method according to claim 17, wherein the copper chelator is a copper I chelator.

23. The method according to claim 17, wherein the copper chelator is N,N,N′

,N′

-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA, neocuproine, N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC), 1,10 phenanthroline, or a derivative thereof, trientine, glutathione, histadine, polyhistadine or tetra-ethylenepolyamine (TEPA).

24. The method according to claim 17, wherein the copper chelator is 1,10 phenanthroline, bathophenanthroline disulfonic acid (4,7odiphenyl-1,10-phenanthroline disulfonic acid) or bathocuproine disulfonic acid (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).

25. The method according to claim 17, wherein the reducing agent is acorbate, Tris(2-Carboxyethyl) Phosphine (TCEP), TCP (2,4,6-trichlorophenol), NADH, NADPH, thiosulfate, 2-mercaptoethanol, dithiothreotol, glutathione, cysteine, metallic copper, quinone, hydroquinone, vitamin K₁, Fe²⁺, Co²⁺, and an applied electric potential.

26. The method according to claim 17, wherein the reducing agent is ascorbate.

27. The method according to claim 17, wherein the separating step comprises chromatography or electrophoresis.

28. The method according to claim 27, wherein the chromatography comprises one or more of FPLC, HPLC, liquid chromatography (LC), size exclusion chromatography, ion exchange chromatography, or affinity chromatography.

29. The method according to claim 27, wherein electrophoresis comprises gel electrophoresis, 1 dimensional (1D) gel electrophoresis, 2 dimensional (2D) gel electrophoresis, native gel electrophoresis, denaturing gel electrophoresis, isoelectric focusing, or capillary electrophoresis.

30. The method according to claim 27, further comprising Western blotting.

31. The method according to claim 17, wherein the azido modified biomoelcule is modified by the metabolic incorporation of azido modified carbohydrates.

32. The method according to claim 17, wherein the azido modified biomolecule is modified by the enzymatic incorporation of azido modified carbohydrates.

39. The method according to claim 30, wherein the solid or semi-solid support is a slide, an array, an agarose gel, a polyacrylamide gel, a hydrogel, a polymeric particle or glass.

33. An azide-alkyne cycloaddition reaction mixture comprising:

a reporter molecule, solid support or carrier molecule that comprises a terminal alkyne moiety;

an azido modified biomoelcule;

copper ions;

at least one reducing agent; and

a copper chelator.

34. The reaction mixture according to claim 33, wherein the copper chelator is a copper(I) chelator.

35. The method according to claim 33, wherein the copper chelator is NNNN-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), EDTA, neocuproine, N-(2-acetamido)iminodiacetic acid (ADA), pyridine-2,6-dicarboxylic acid (PDA), S-carboxymethyl-L-cysteine (SCMC), 1,10 phenanthroline, or a derivative thereof, trientine, or tetra-ethylenepolyamine (TEPA).

36. The method according to claim 33, wherein the copper chelator is 1,10 phenanthroline, bathophenanthroline disulfonic acid (4,7odiphenyl-1,10-phenanthroline disulfonic acid) or bathocuproine disulfonic acid (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate).

37. An azide-alkyne cycloaddition reaction mixture comprising:

a reporter molecule, solid support or carrier molecule that comprises an azido moiety;

a biomoelcule comprising a terminal alkyne;

copper ions;

at least one reducing agent; and

a copper chelator.

38. A method for detecting immobilized azido modified biomolecules, wherein the method comprises:

a. immobilizing the azido modified biomolecules on a solid or semi-solid matrix to form an immobilized azido modified biomoelcule;

b. contacting the immobilized azido modified biomoelcule with a reporter molecule that contains an azide reactive group to form a contacted azido modified biomoelcule;

c. incubating the contacted azido modified biomoelcule for a sufficient amount of time to form a reporter molecule-biomoelcule conjugate;

d. illuminating the reporter molecule-biomoelcule conjugate with an appropriate wavelength to form an illuminated reporter molecule-biomoelcule conjugate;

e. observing the illuminated reporter molecule-biomoelcule conjugate whereby the immobilized azido modified biomolecule is detected.

40. A kit comprising:

UDP-GalNAz;

a GalT enzyme;

an azide reactive reporter molecule, carrier molecule or solid support.