Labeling and detection of post translationally modified proteins

• US 9,645,140 B2
• Filed: 07/14/2014
• Issued: 05/09/2017
• Est. Priority Date: 02/10/2006
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Agnew, Brian, Gee, Kyle, Nyberg, Tamara
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US14/330,727
• Publication Number:
  US 20150051096A1
• Time in Patent Office: 1,030 Days
• 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 detecting an azido modified protein or peptide, comprising:

a) forming an azide-alkyne cycloaddition reaction mixture by adding the followings in a reaction mixture;

a reporter molecule that comprises a terminal alkyne moiety;

the azido modified protein or peptide;

copper (II) ions;

at least one reducing agent; and

a copper chelator selected from the group consisting of 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), trientine, glutathione, histadine, polyhistadine or tetra-ehylenepolyamine (TEPA), 1,10 phenanthroline, bathophenanthroline disulfonic acid (4,7-diphenyl-1,10-phenanthroline disulfonic acid) and bathocuproine disulfonic acid (2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate), wherein the copper chelator is added to the reaction mixture after the copper (II) ions have been contacted with the at least one reducing agent, and wherein the at least on reducing agent is selected from the group consisting of 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⁺, and Co²⁺;

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

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

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

e) observing the illuminated protein or peptide-reporter molecule conjugate wherein the azido modified protein or peptide is detected.

2. The method according to claim 1, wherein the protein is a glycoprotein.

3. The method according to claim 1, 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, trientine, glutathione, histadine, polyhistadine or tetra-ethylenepolyamine (TEPA).

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

5. The method according to claim 1, wherein the reducing agent is ascorbate.

6. The method according to claim 1, wherein the separating step comprises chromatography or electrophoresis.

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

8. The method according to claim 1, wherein the reporter molecule is an enzyme substrate, a fluorescent protein, a particle, or a hapten.