Detecting targets using mass tags and mass spectrometry

• US 9,291,597 B2
• Filed: 07/01/2011
• Issued: 03/22/2016
• Est. Priority Date: 07/02/2010
• Status: Active Grant

• Current Assignee: Ventana Medical Systems Incorporated (Roche Holding AG)
• Original Assignee: Ventana Medical Systems Incorporated (Roche Holding AG)
• Inventors: Hong, Rui, Wang, Hong, Lefever, Mark, Froehlich, Jan, Bieniarz, Christopher, Kelly, Brian, Miller, Phillip
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US13/805,983
• Publication Number:
  US 20130122516A1
• Time in Patent Office: 1,726 Days
• US Class Current: 1/1
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

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.

Claims

1. A method, comprising:

(a) providing a formalin-fixed, paraffin-embedded tissue sample comprising a plurality of targets;

(b) contacting the 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;

(c) 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;

(d) contacting the tissue sample with a first enzyme substrate moiety and separately a first mass tag precursor, wherein the first enzyme substrate moiety reacts with the first enzyme and the first mass tag precursor to produce and deposit a first mass tag at the first target;

(e) deactivating the first enzyme;

(f) 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;

(g) contacting the tissue sample with a second enzyme substrate moiety and separately a second mass tag precursor, wherein the second enzyme substrate moiety reacts with the second enzyme and the second mass tag precursor to produce and deposit a second mass tag at the second target, wherein the first and second enzyme substrate moieties have the same chemical structure, the first and second mass tag precursors have the same chemical structure and at least one of the first mass tag precursor or second mass tag precursor is isotopically labeled such that the first mass tag and the second mass tag differ in mass;

(h) ionizing the first mass tag and the second 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;

(i) detecting each mass code using a mass spectrometer; and

(j) 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;

wherein the first mass tag precursor and the second mass tag precursor are selected from the group consisting of

2. The method of claim 1, wherein the first mass tag precursor and the second mass tag precursor are selected from the group consisting of

3. The method of claim 1, wherein the first and second enzyme substrate moieties are selected from a methoxy naphthol or naphthol-azo compound and the first enzyme and the second enzymes are phosphatase enzymes.

4. The method of claim 1, 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 enzyme substrate moiety and an additional mass tag precursor, wherein the additional enzyme substrate moiety reacts with the additional enzyme and the additional mass tag precursor to produce and deposit an additional mass tag at the one of the one or more subsequent targets, wherein the additional enzyme substrate moiety has the same chemical structure as the first and second enzyme substrate moieties, the additional mass tag precursor has the same chemical structures as the first and second mass tag precursors, and the additional 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.

5. The method of claim 1, wherein, the first enzyme substrate moiety, the second enzyme substrate moiety, and any additional enzyme substrate moieties have the structure

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

7. The method of claim 6, wherein the cancer biomarkers comprise at least two breast cancer biomarkers selected from the group consisting of ER, PR, Her1, Her2, Her3, Her4, and Ki67.

8. The method of claim 6, wherein each cancer biomarker comprises a protein or a peptide, and wherein the first specific binding moiety is an antibody capable of recognizing and binding to a first cancer biomarker, the second specific binding moiety is an antibody capable of recognizing and binding to a second cancer biomarker, and each subsequent specific binding moiety is an antibody capable of recognizing and binding to a subsequent cancer biomarker.

9. The method of claim 6, wherein each cancer biomarker comprises a nucleic acid sequence, and wherein the first specific binding moiety is a labeled probe capable of recognizing and hybridizing to a first cancer biomarker or a portion thereof, the second specific binding moiety is a labeled probe capable of recognizing and hybridizing to a second cancer biomarker or a portion thereof, and each subsequent specific binding moiety is a labeled probe capable of recognizing and hybridizing to a subsequent cancer biomarker or a portion thereof.

10. The method of claim 1, wherein the first target comprises an intact form of a biomarker and the second target comprises one or more truncated forms of the biomarker.

11. The method of claim 10, wherein the first specific binding moiety is capable of recognizing and binding to the intact form of the biomarker, and the second specific binding moiety is capable of recognizing and binding to a truncated form of the biomarker and to the intact form of the biomarker, wherein the second enzyme substrate moiety reacts with the second enzyme and the second mass tag precursor to produce and deposit a second mass tag at the truncated form of the biomarker and at the intact form of the biomarker, and wherein quantifying the truncated form of the biomarker comprises quantifying the first mass code and the second mass code and determining the numerical difference between the quantified second mass code and the quantified first mass code.

12. A method, comprising:

(a) providing a formalin-fixed, paraffin-embedded tissue sample comprising a plurality of targets;

(b) contacting the 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;

(c) 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;

(d) contacting the tissue sample with a first enzyme substrate moiety and separately a first mass tag precursor, wherein the first enzyme substrate moiety reacts with the first enzyme and the first mass tag precursor to produce and deposit a first mass tag at the first target;

(e) deactivating the first enzyme;

(f) 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;

(g) contacting the tissue sample with a second enzyme substrate moiety and separately a second mass tag precursor, wherein the second enzyme substrate moiety reacts with the second enzyme and the second mass tag precursor to produce and deposit a second mass tag at the second target, wherein the first and second enzyme substrate moieties have the same chemical structure, the first and second mass tag precursors have the same chemical structure and at least one of the first mass tag precursor or second mass tag precursor is isotopically labeled such that the first mass tag and the second mass tag differ in mass;

(h) ionizing the first mass tag and the second 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;

(i) detecting each mass code using a mass spectrometer; and

(j) 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;

wherein the tissue sample comprises from 2 to 4 targets, and the first mass tag precursor, the second mass tag precursor are selected from the group consisting of

13. The method of claim 12, wherein the first mass tag precursor, the second mass tag precursor, and any additional mass tag precursors are selected from the group consisting of