Detecting targets using mass tags and mass spectrometry

摘要

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.

主权项

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 ofwhere n is 1, 2, 3, 4, or 5 and wherein D is deuterium.