Detecting targets using mass tags and mass spectrometry

Patent metadata

Field	Value
Publication number	US-10078083-B2
Application number	US-201514981181-A
Country	US
Kind code	B2
Filing date	Dec 28, 2015
Priority date	Jul 2, 2010
Publication date	Sep 18, 2018
Grant date	Sep 18, 2018

Abstract

Particular disclosed embodiments disclosed herein concern using a one or more various mass tags, which can be specifically deposited at targets through direct or indirect enzymatic-catalyzed transformation, to provide a method for identifying targets in tissue samples. The mass tags may be labeled with stable isotopes to produce mass tags having the same chemical structure but different masses. Mass codes produced by ionizing the mass tags are detected and/or quantified using mass spectrometry. The method can be used for multiplexed detection of multiple targets in a particular sample. In some embodiments, a map divided into sections representing sections of the tissue sample may be prepared, with the map sections including data corresponding to quantification data wherein the size of a mass peak is determined and correlated with the amount of a target for the corresponding tissue sample section.

First claim

We claim:

1. A method of analyzing a sample comprising:
contacting the sample with a specific binding moiety selected to recognize a target in the sample,
localizing an enzyme proximally to the target,
contacting the enzyme with a water soluble enzyme substrate to produce and deposit an insoluble mass tag precipitate proximal to the target, and
detecting the mass tag using mass spectrometry,
wherein the water soluble enzyme substrate is selected from the group consisting of a tyramine or a tyramide derivative.

2. The method according to claim 1 , wherein the sample further includes a second target.

3. The method according to claim 2 , further comprising producing and depositing a second mass tag proximal to the second target.

4. The method according to claim 3 , wherein the second mass tag and the first mass tag have equivalent structures but different masses.

5. The method according to claim 3 , wherein the second mass tag and the first mass tag differ by one or more hydrogen to deuterium substitution.

6. The method of claim 1 , wherein the enzyme substrate is a mass tag precursor conjugate having the formula Enzyme Substrate Moiety-(Optional Linker)-Mass Tag Precursor.

7. The method of claim 6 , wherein the mass tag precursor conjugate is water soluble.

8. The method of claim 1 , wherein the enzyme substrate is a mass tag precursor conjugate having the formula

where R 16 is selected from ether, hydroxyl, and -(nitrogen-R 11 )—, where R 11 is selected from aliphatic, heteroaliphatic, aryl, heteroaryl, and hydrogen, R 17 is selected from aliphatic, heteroaliphatic, aryl, and heteroaryl, n is 1-20, Y is selected from oxygen, sulfur, and -(nitrogen-R 11 )—; the optional linker, if present, is selected from aliphatic, heteroaliphatic, or heterobifunctional linkers; the optional carrier, if present, is a polymer, a biomolecule, a liposome, a micelle, or a nanoparticle; and the mass tag is a moiety capable of producing a mass code upon cleavage and ionization of the conjugate,
wherein

is a substrate for the enzyme.

9. The method of claim 1 , wherein the enzyme is selected from the group consisting of horseradish peroxidase and alkaline phosphatase.

10. The method of claim 1 , wherein the plurality of targets comprises a plurality of cancer biomarkers.

11. The method of claim 10 , wherein the cancer biomarkers comprise at least two breast cancer biomarkers selected from ER, PR, Her1, Her2, Her3, Her4, or Ki67.

12. A method, comprising:
(a) contacting a tissue sample with a first specific binding moiety capable of recognizing and binding to a first target in the tissue sample and a second specific binding moiety capable of recognizing and binding to a second target in the tissue sample;
(b) contacting the tissue sample with a first conjugate comprising a first enzyme and a first antibody capable of recognizing and binding to the first specific binding moiety;
(c) contacting the tissue sample with a first water soluble enzyme substrate moiety and a first water soluble mass tag precursor, wherein the first water soluble enzyme substrate moiety reacts with the first water soluble enzyme and the first water soluble mass tag precursor to produce and deposit a first insoluble mass tag at the first target;
(d) deactivating the first enzyme;
(e) contacting the tissue sample with a second conjugate comprising a second enzyme and a second antibody capable of recognizing and binding to the second specific binding moiety;
(f) contacting the tissue sample with a second water soluble enzyme substrate moiety and a second water soluble mass tag precursor, wherein the second water soluble enzyme substrate moiety reacts with the second water soluble enzyme and the second water soluble mass tag precursor to produce and deposit a second insoluble mass tag at the second target, wherein the first and second enzyme water soluble substrate moieties have the same chemical structure, the first and second water soluble mass tag precursors have the same chemical structure and at least one of the first water soluble mass tag precursor or second water soluble mass tag precursor is isotopically labeled such that the first insoluble mass tag and the second insoluble mass tag differ in mass;
(g) ionizing the first insoluble mass tag and the second insoluble mass tag to produce a first mass code and a second mass code, wherein each mass code has the same chemical structure but differs in mass from any other mass code produced from any other mass tag;
(h) detecting each mass code using a mass spectrometer; and
(i) quantifying relatively each target by quantifying an amount of each mass code by measuring a mass spectrometric peak having a m/z ratio corresponding to an expected m/z ratio of that mass code.

13. The method of claim 12 , wherein the first water soluble mass tag precursor and the second mass tag precursor have the structures

where n is 1, 2, 3, 4, or 5.

14. The method of claim 13 , wherein the first water soluble mass tag precursor and the second mass tag precursor have the structures

15. The method of claim 12 , wherein the first and second water soluble enzyme substrate moieties are selected from the group consisting of a methoxy naphthol or naphthol-azo compound and the first enzyme and the second enzymes are phosphatase enzymes.

16. The method of claim 12 , further comprising:
contacting the tissue sample in step (b) with one or more subsequent specific binding moieties, each subsequent specific binding moiety capable of recognizing and binding to a subsequent target in the tissue sample;

deactivating the second enzyme after step (g);
sequentially performing the following steps for each of the one or more subsequent targets before performing step (h):
contacting the tissue sample with an additional conjugate for one of the one or more subsequent targets, the additional conjugate comprising an additional antibody and an additional enzyme, wherein the additional antibody is capable of recognizing and binding to the one of the one or more subsequent targets or to a specific binding moiety previously bound to the one of the one or more subsequent targets,
contacting the sample with an additional water soluble enzyme substrate moiety and an additional water soluble mass tag precursor, wherein the additional water soluble enzyme substrate moiety reacts with the additional water soluble enzyme and the additional mass tag precursor to produce and deposit an additional insoluble mass tag at the one of the one or more subsequent targets, wherein the additional water soluble enzyme substrate moiety has the same chemical structure as the first and water soluble second enzyme substrate moieties, the additional water soluble mass tag precursor has the same chemical structures as the first and second water soluble mass tag precursors, and the additional insoluble mass tag precursor is isotopically labeled such that the additional mass tag has a mass that differs from the masses of the first mass tag, the second mass tag, and any other additional mass tag; and
deactivating the additional enzyme if another subsequent target is present.

17. The method of claim 1

Assignees

• Ventana Med Syst Inc

Inventors

• Hong Rui
• Wang Hong
• Lefever Mark
• Froehlich Jan
• Bieniarz Christopher
• Kelly Brian Daniel
• Miller Phillip

Classifications

• G01N27/62

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• G01N33/6848 Primary

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