Methods and compositions for analyzing proteins

• US 7,255,999 B2
• Filed: 05/21/2002
• Issued: 08/14/2007
• Est. Priority Date: 05/21/2001
• Status: Active Grant

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Salimi-Moosavi, Hossein, Hernandez, Vincent S., Tahir, Syed Hasan, Wallweber, Gerald J., Kirakossian, Hrair, Matray, Tracy J.
• Primary Examiner(s): Le; Long V.
• Assistant Examiner(s): Haq; Shafiqul
• Application Number:
  US10/154,042
• Publication Number:
  US 20030013126A1
• Time in Patent Office: 1,911 Days
• Field of Search: 435/7.1, 435/7.92, 435/7.72, 435/7.95, 435/21, 436/544, 436/546, 436/161, 436/824, 530/388.9, 530/402, 530/412
• US Class Current: 435/7.1
• 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: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins

Abstract

Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone a post-translational modification. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and a first binding agent for a binding site on a target polypeptide is subjected to conditions under which binding of respective binding moieties occurs. The binding site is the result of post-translational modification activity involving the target polypeptide. The method may be employed to determine the target polypeptide itself. In another embodiment the presence and/or amount of the target polypeptide is related to the presence and/or amount and/or activity of an agent such as an enzyme involved in the post-translational modification of the target polypeptide. The interaction between the first binding agent and the binding site brings the cleavage-inducing moiety into close proximity to a cleavable moiety, which is associated with the polypeptide and is susceptible to cleavage only when in proximity to the cleavage-inducing moiety. In this way, an electrophoretic tag for each of the polypeptides may be released. Released electrophoretic tags are separated and the presence and/or amount of the target polypeptides are determined based on the corresponding electrophoretic tags.

Claims

1. A method for determining the presence or absence of one or more target polypeptides in a sample suspected of containing the target polypeptides, the method comprising the steps of:

(a) mixing the sample with (i) a class-specific reagent comprising, a cleavage-inducing moiety attached a first binding agent that specifically bind a post-translational modification on the one or more target polypeptides, and (ii) one or more electrophoretic probes, each of which is attached to a first binding moiety that specifically binds one of the target polypeptides and one or more electrophoretic tags each attached thereto by a cleavable linkage, under conditions that permit the class-specific reagent and the one or more electrophoretic probes to bind to the one or more target polypeptides, and which brings the cleavage inducing moiety within an effective proximity to a cleavable linkage of said one or more electrophoretic probe;

(b) cleaving the cleavable linkages(s) of any of the one or more electrophoretic probes within the effective proximity of the cleavage-inducing moiety so that one or more electrophoretic tags are released wherein cleaving is by the cleavage-inducing moiety; and

(c) determining the presence or absence of the one or more target polypeptides based on the presence or absence of the released electrophoretic tags.

2. The method of claim 1 wherein said one or more target polypeptides is a plurality of target polypeptides.

3. The method of claim 2 wherein said cleavage-inducing moiety is a sensitizer that produces an active species.

4. The method of claim 3 wherein said sensitizer is a photosensitizer, said cleavable linkage is an oxidation-labile linkage, and said active species is selected from the group consisting of hydrogen peroxide, hydroxyl radical, superoxide anion, phenoxy radical, and singlet oxygen.

5. The method of claim 4 wherein said active species is singlet oxygen.

6. The method of claim 5 wherein said binding agent of said class-specific reagent is selected from the group consisting of antibodies, protein receptors, ligands of protein receptors, lectins, biotin-containing moieties, boronic acid-containing moieties, aptamers, enzyme substrates, enzyme cofactors, and enzyme subunits, and wherein said binding moiety of said electrophoretic probe is an antibody or an antibody binding composition.

7. The method of claim 5 wherein said post-translational modification is selected from the group consisting of phosphorylation, glycosylation, ribosylation, acetylation, acylation, methylation, lipidation, isoprenylation and ubiquitination.

8. The method of claim 5 wherein said plurality of target polypeptides is in the range of from 2 to 100.

9. The method of claim 8 wherein said plurality of target polypeptides is in the range of from 5 to 50.

10. The method of claim 5 wherein said photosensitizer is selected from the group consisting of porphyrins, phthalocyanines, halogenated derivatives of a fluorescein dye, halogenated derivatives of a rhodamine dye, and naphthalocyanines.

11. The method of claim 5 wherein said cleavable linkage comprises an olefin, a thioether, a selenoether, a thiazole, an oxazole, or an imidazole.

12. The method of claim 5 wherein said electrophoretic probe is defined by the following formula:

T-(L-L)_k wherein;

T is a binding moiety specific for a target polypeptide, L is an oxidation-labile linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1.

13. The method of claim 12 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

14. The method of claim 13 wherein E is a fluorescent, water-soluble organic compound having a molecular weight in the range of from about 150 to 2500 daltons.

15. The method of claim 13 wherein E has the form (M,D), wherein D is a fluorescent dye and wherein M is a mobility modifying moiety that is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

16. The method of claim 12 wherein said electrophoretic tag, E, is defined by the formula:

A—

M—

D

wherein;

A is —

C(O)R, where R is aliphatic, aromatic, alicyclic or heterocyclic having from 1 to 8 carbon atoms and 0 to 4 heteroatoms selected from the group consisting of O, S. and N;

—

CH₂—

C(═

O)—

NH—

CHO;

—

SO₂H;

—

CH₂—

C(═

O)O—

CHO;

—

C(═

O)NH—

(CH₂)_n—

NH—

C(═

O)C(═

O)—

(C₆H₅)—

, where n is in the range of from 2 to 12;

D is a fluorescent dye; and

M is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur, with the proviso that the total molecular weight of E be within the range of from about 150 to about 5000 daltons.

17. The method of claim 16 wherein D is a fluorescein dye.