Compositions and methods for detection and isolation of phosphorylated molecules

• US 7,776,533 B2
• Filed: 10/24/2006
• Issued: 08/17/2010
• Est. Priority Date: 05/03/2002
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Patton, Wayne F, Haugland, Richard P, Steinberg, Thomas H, Gee, Kyle R, Beechem, Joseph, Agnew, Brian
• Primary Examiner(s): CHEU, CHANGHWA J
• Application Number:
  US11/552,275
• Publication Number:
  US 20070054304A1
• Time in Patent Office: 1,393 Days
• Field of Search: 435/4, 435/21, 435/17, 436/103, 436/104, 436/105
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2333/4709 Amyloid plaque core protein
• 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 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: Y10T 436/142222 Hetero-O [e.g., ascorbic acid, etc.]
• CPC Class Codes: Y10T 436/145555 Hetero-N
• CPC Class Codes: Y10T 436/147777 Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
• CPC Class Codes: Y10T 436/201666 Carboxylic acid

Abstract

The present invention relates to phosphate-binding compounds that find use in binding, detecting and isolating phosphorylated target molecules including the subsequent identification of target molecules that interact with phosphorylated target molecules or molecules capable of being phosphorylated. A binding solution is provide that comprises a phosphate-binding compound, an acid and a metal ion wherein the metal ion simultaneously interacts with an exposed phosphate group on a target molecule and the metal chelating moiety of the phosphate-binding compound forming a bridge between the phosphate-binding compound and a phosphorylated target molecule resulting in a ternary complex. The binding solution of the present invention finds use in binding and detecting immobilized and solubilized phosphorylated target molecules, isolation of phosphorylated target molecules from a complex mixture and aiding in proteomic analysis wherein kinase and phosphatase substrates and enzymes can be identified.

Claims

1. A method for detecting a phosphorylated target molecule in a sample immobilized on a membrane, said method comprising the steps of:

i) immobilizing said sample to a membrane;

ii) contacting said membrane of step i) with a binding solution, wherein said binding solution comprises;

a) phosphate-binding compound having formula (A)m(L)n(B) wherein A is a detectable chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4;

b) a salt comprising metal ions; and

c) an acid;

iii) incubating said membrane of step ii) and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule; and

,

iv) detecting said phosphate-binding compound whereby said phosphorylated target molecule is detected,

wherein step iii) further comprises contacting said membrane with a wash solution following contact with said binding solution, wherein said phosphorylated target molecule is selected from the group consisting of proteins, peptides, and nucleotides and wherein B of said formula (A)m(L)n(B) comprises a compound selected from the group consisting of Compound 1, 2, 3, 4, 5, 9, 10, 11 and 12 of Table 2.

2. The method according to claim 1, further comprising adding an additional detection reagent to said membrane.

3. The method according to claim 2, wherein said additional detection reagent is a staining solution specific for total proteins, a staining solution specific for glycoproteins or an antibody.

4. A method for detecting a phosphorylated target molecule in a sample immobilized on a gel, said method comprising the steps of:

i) immobilizing said sample on a gel;

ii) contacting said gel of step i) with a binding solution, wherein said binding solution comprises;

a) a phosphate-binding compound having formula (A)m(L)n(B) wherein A is a detectable chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4;

b) a salt comprising metal ions; and

,

c) an acid;

iii) incubating said gel of step ii) and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule; and

,

iv) detecting said phosphate-binding compound whereby said phosphorylated target molecule is detected,

wherein step iii) further comprises contacting said gel with a wash solution following contact with said binding solution, wherein said phosphorylated target molecules are selected from the group consisting of proteins, peptides, and nucleotides, wherein B of said formula (A)m(L)n(B) comprises a compound selected from the group consisting of Compound 1, 2, 3, 4, 5, 9, 10, 11 and 12 of Table 2.

5. A method for detecting a phosphorylated target molecule in a sample immobilized on a matrix, said method comprising the steps of:

i) immobilizing said sample on an matrix;

ii) contacting said matrix of step i) with a binding solution, wherein said binding solution comprises;

a) a phosphate-binding compound having formula (A)m(L)n(B) wherein A is a detectable chemical moiety, L is a linker, B is a metal-chelating moiety, m is an integer from 1 to 4 and n is an integer from 0 to 4;

b) a salt comprising metal ions; and

,

c) an acid;

iii) incubating said matrix of step ii) and said binding solution for sufficient time to allow said phosphate-binding compound to associate with said phosphorylated target molecule; and

,

iv) detecting said phosphate-binding compound whereby said phosphorylated target molecule is detected,

wherein said sample is selected from the group consisting of proteins, peptides, nucleotides, lipids, kinase substrates, phosphatase substrates, phosphate binding proteins and carbohydrates, and wherein said matrix is a polymeric gel, glass, polymeric membrane, plastic, polymeric microparticle, wherein B of said formula (A)m(L)n(B) comprises a compound selected from the group consisting of Compound 1, 2, 3, 4, 5, 9, 10, 11 and 12 of Table 2.

6. The method according to claim 5, wherein said step iii) further comprises contacting said matrix with a wash solution following contact with said binding solution.

7. The method according to claim 6, further comprising adding an additional detection reagent to said matrix.

8. The method according to claim 6, wherein said additional detection reagent is a staining solution specific for total proteins, a staining solution specific for glycoproteins or an antibody.