In vivo proteomic mapping

Patent metadata

Field	Value
Publication number	US-9624524-B2
Application number	US-201214125819-A
Country	US
Kind code	B2
Filing date	Jun 15, 2012
Priority date	Jun 15, 2011
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

Abstract

The application discloses methods, materials, and compositions for the labeling of molecules, for example, proteins, in living cells or in subcellular compartments of living cells. In particular, the application relates to proteomic analysis methods; materials and compositions and means based on direct tagging of unknown proteins with tagging enzymes (such as biotin ligase or a peroxidase) within the vicinity of a tagging substrate (such as a tyramide) within living cells, with optional targeting to specific subcellular locations by expression of genetic constructs.

First claim

What is claimed is:

1. A method for proteomic mapping of a living cell comprising:
contacting a living cell with a tagging enzyme under physiological conditions wherein the tagging enzyme catalyzes a reaction with a tagging substrate resulting in the tagging of proteins within an intracellular spatial location around the enzyme and/or substrate;
wherein the tagging enzyme is genetically targeted or is targeted using a targeting sequence such as antibody to a subcellular region; and
isolating and analyzing the tagged proteins to create a first proteome map.

2. The method of claim 1 , further comprising contacting the living cell with a tagging substrate.

3. The method of claim 1 , wherein a nucleic acid construct encoding the tagging enzyme is delivered to the cell.

4. The method of claim 1 , wherein the living cell is exposed to a therapeutic agent prior to or during the step of contacting with a tagging enzyme.

5. The method of claim 4 , further comprising contacting a second living cell exposed to a second therapeutic agent with a tagging enzyme under physiological conditions wherein the tagging enzyme catalyzes a reaction with a tagging substrate resulting in the tagging of proteins within an intracellular spatial location around the enzyme and/or substrate;
wherein the tagging enzyme is genetically targeted to a subcellular region; and
isolating and analyzing the tagged proteins to create a second proteomic map.

6. The method of claim 5 , wherein the first and second proteome maps are compared.

7. A method of mapping the proteome of a living cells comprising:
contacting a living cell with a tagging enzyme and a tagging substrate in order to catalyze a reaction resulting in the production of a tagged protein within the spatial location around the tagging enzyme and/or substrate of the tagging enzyme, and further comprising generating a proteome map based on the tagged proteins;
wherein the tagging enzyme is genetically targeted to a subcellular region.

8. The method of claim 1 , wherein the tagging enzyme is a peroxidase.

9. The method of claim 1 , wherein the tagging enzyme is HRP.

10. The method of claim 1 , wherein the tagging substrate is a tyramide.

11. The method of claim 1 , wherein the tagging substrate is a labeled tyramide.

12. The method of claim 1 , wherein the tagging substrate is a biotinylated tyramide.

13. The method of claim 7 , further comprising isolating the tagged proteins.

14. The method of claim 13 , further comprising analyzing the isolated proteins.

15. A method for proteomic mapping, comprising
contacting a living cell with a tagging enzyme under physiological conditions wherein the tagging enzyme is targeted to a spatial location of the cell and catalyzes a reaction with a tagging substrate resulting in the tagging of proteins within the spatial location around the tagging enzyme and/or substrate of the tagging enzyme, and
isolating and analyzing the tagged proteins to create a first proteome map, wherein the proteome map is a map of a proteome which is a set of expressed proteins expressed in a cell, a subcellular compartment or an organism under a set of conditions.

16. The method of claim 15 , wherein a nucleic acid construct encoding the tagging enzyme is delivered to the cell.

Assignees

• Ting Alice Y
• Martell Jeffrey Daniel
• Rhee Hyun-Woo
• Zou Peng
• Massachusetts Inst Technology

Inventors

• Ting Alice Y
• Martell Jeffrey Daniel
• Rhee Hyun-Woo
• Zou Peng

Classifications

• G01N33/6803

  General methods of protein analysis not limited to specific proteins or families of proteins · CPC title

• G01N33/5023

  on expression patterns · CPC title

• C12Q1/28 Primary

  involving peroxidase · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title