Proteome-wide quantification of small molecule binding to cellular target proteins

Patent metadata

Field	Value
Publication number	US-8959984-B2
Application number	US-68146808-A
Country	US
Kind code	B2
Filing date	Sep 26, 2008
Priority date	Oct 5, 2007
Publication date	Feb 24, 2015
Grant date	Feb 24, 2015

Abstract

This invention relates to methods for the evaluation and/or quantification of the binding affinity of small molecules or other compounds to target components contained within an analyte, such as target proteins contained within the proteome of a cell or tissue.

First claim

The invention claimed is:

1. A method for evaluating a binding affinity of a target component of an analyte to a compound, wherein the target component is a protein, wherein the compound is a kinase inhibitor, and wherein the analyte is selected from the group consisting of a proteome, a mixture of different proteomes, a cell extract or lysate, a tissue extract or lysate, a cell culture supernatant, a tissue culture supernatant or a body fluid, the method comprising
(a) contacting a first aliquot of the analyte with a first solid support onto which the compound is immobilized in a defined concentration under conditions permitting the binding of the target component in the analyte to the immobilized compound, wherein the target component is labelled with a detectable label, and wherein the detectable label is different in each aliquot of the analyte;
(b) contacting a second aliquot of the analyte with a solid support onto which the compound is immobilized as in step (a), wherein said solid support comprises the same solid support material as the solid support in step (a), and wherein the compound is immobilized in the same concentration as in step (a) under conditions permitting the binding of the target component to the compound, and subsequently separating a supernatant of the second aliquot of the analyte from said solid support;
(c) contacting the supernatant separated from the solid support in step (b) with a solid support onto which the compound is immobilized as in step a), wherein said solid support comprises the same solid support material as the solid support in step a) and wherein the compound is immobilized in the same concentration as in step a) under conditions permitting the binding of the target component to the compound;
(d) determining the amounts of the target component bound to the solid support in step (a) and the solid support in step (c);
(e) comparing the amount of target component bound to the solid support in step (a) to the amount of target component bound to the solid support in step (c); and
(f) determining the K d value of the binding of the target component to the immobilized compound based on the comparison in step (e).

2. The method of claim 1 , further comprising contacting a third aliquot of said analyte with a solid support, wherein the solid support comprises the same solid support material as the solid support in step (a) but does not have said compound immobilized onto it, and determining the amount of target component bound to said solid support;
wherein step (e) further comprises comparing the amounts of target component bound to the solid support in step (a) and the solid support in step (c) to the amount of target component bound to said solid support that does not have said compound immobilized onto it.

3. A method for evaluating a binding affinity of a target component of an analyte to a compound, wherein the target component is a protein, wherein the compound is a kinase inhibitor, and wherein the analyte is selected from the group consisting of a proteome, a mixture of different proteomes, a cell extract or lysate, a tissue extract or lysate, a cell culture supernatant, a tissue culture supernatant or a body fluid, the method comprising
(a) contacting a first aliquot of the analyte with a first solid support onto which the compound is immobilized under conditions permitting the binding of the target component in the analyte to the immobilized compound, wherein the target component is labelled with a detectable label, and wherein the detectable label is different in each aliquot of the analyte;
(b) contacting a second aliquot of the analyte with a second solid support, wherein the second solid support is the same as the first solid support except said compound is immobilized onto the second solid support at a higher concentration than on the first solid support of step (a) under conditions permitting the binding of the target component to the immobilized compound;
(c) determining the amounts of target component bound to the first solid support in step (a) and the second solid support in step (b); and
(d) comparing the amounts of target component bound to the first solid support in step (a) and the second solid support in step (b); and
(e) determining the K d value of the binding of the target component to the compound based on the comparison in step (d).

4. The method of claim 3 , wherein a ratio of the concentrations in which the compound is immobilized onto the solid support in steps (a) and (b) is selected from the ratios of about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.

5. The method of claim 3 , further comprising contacting a third aliquot of said analyte with a third solid support, wherein the third solid support does not have said compound immobilized onto it, and determining the amount of target component bound to said third solid support;
wherein step (d) further comprises comparing the amounts of target component bound to the first solid support in step (a) and the second solid support in step (b), to the amount of target component bound to said third solid support.

6. The method of claim 1 , wherein said labelling is done by a stable isotope labelling with amino acids in cell culture (SILAC) procedure, and wherein said determination according to step (d) is done by quantitative protein analysis via mass spectrometry.

7. The method of claim 1 , wherein the determining step (d) comprises combining said bound target components into one sample and detecting the amounts of differently labelled target components in said sample.

8. The method of claim 7 , farther comprising a step of eluting the target components from the solid supports in steps (a) and (c) prior to being combined into said sample.

9. The method of claim 1 , wherein the compound is present during said contacting steps in a molar excess compared to the target component.

10. The method of claim 1 , wherein said contacting steps (a) and (b) are performed simultaneously.

11. The method according to claim 1 , wherein the proteome is present in or derived from one single cell or cell culture, a mixture of cells, a tissue, an organ or an organism.

12. The method according to claim 1 , wherein the solid support is selected from the group consisting of filters, glass slides, silicon surfaces, beads and a customized chemical microarray.

13. The method of claim 12 , wherein said beads are sepharose beads, optionally epoxy-activated or NHS-activated, or agarose beads.

14. The method according to claim 1 , wherein the immobilized compound is immobilized onto the solid support via adsorption, absorption, ionic bonding, covalent bonding, an amino-group or carboxy-group or hydroxy-group, (strept)avidin-biotin, or thiol-gold interactions.

15. The method according to claim 1 , wherein said label is selected from the group consisting of radiolabels, dye labels, labels that can be detected with antibodies, enzyme labels, phosphorescent markers, fluorescent markers, chemiluminescent markers, phosphatases, avidin, streptavidin, biotin, TAP, peroxidases and labels having a detectable mass.

16. The method according to claim 1 , wherein said contacting is performed essentially under physiological conditions.

17. The method according to claim 1 , wherein said contacting is performed using a buffer and, optionally, a cofactor selected from NAD+/NADH, cGMP, NADP+/NADPH, ATP, ADP, or cAMP.

18. The method according to claim 1 , wherein the determination according to step (d) comprises detecting the label by a met

Assignees

• Daub Henrik
• Bairlein Michaela
• Sharma Kirti
• Godl Klaus
• Tebbe Andreas
• Weber Christoph
• Max Planck Gesellschaft

Inventors

• Daub Henrik
• Bairlein Michaela
• Sharma Kirti
• Godl Klaus
• Tebbe Andreas
• Weber Christoph

Classifications

• G01N33/54306 Primary

  Solid-phase reaction mechanisms · CPC title

• C12Q1/485

  involving kinase · CPC title

• G01N33/6851

  Methods of protein analysis involving laser desorption ionisation mass spectrometry · CPC title

• G01N30/6043

  in parallel · CPC title

• G01N30/7233

  interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04) · CPC title