Chemical Probe Protein Activity Mapping With Proximity Oligo Detection
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Solution Overview
Problem
Current proteomic methods fail to provide information on protein function, are limited by sample amount, biased towards high abundance proteins, and lose spatial information due to sample homogenization, lacking the ability to measure low abundance proteins and proteins in their natural environment.
Innovation Solution
A novel platform for spatially detecting enzyme activity using molecular constructs with targeting groups and retrieval tags, enabling quantification of protein activity, visualization of localized enzyme activity, and amplification for low abundance proteins through proximity ligation and annealing of oligos.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If gel-based or mass spectrometry-based proteomic experiments are used, then protein abundance information can be obtained, but the amount of sample required is significant and spatial information is lost
Solution Approach 1:
The invention segments the detection process into two distinct phases: (1) in vivo labeling of active proteins with chemical probes within intact tissue sections to preserve spatial information, and (2) extraction and analysis of labeled proteins. This segmentation allows spatial information to be preserved during labeling while enabling sufficient sample extraction for analysis.
Solution Approach 2:
The invention performs preliminary labeling of proteins with chemical probes in situ within tissue sections before extraction. This preliminary action tags proteins with detectable markers while they are still in their native spatial context, allowing subsequent extraction and analysis without losing spatial information.
2Loss of information
If standard proteomic methods are used, then protein presence and abundance can be detected, but information on protein functional state is not provided
Solution Approach 1:
The invention introduces chemical probes as intermediary molecules that specifically bind to active proteins. These probes act as mediators between the protein's functional state and the detection system, transferring information about enzymatic activity to a detectable signal without requiring complex functional assays.
Solution Approach 2:
The invention replaces complex mechanical or biochemical functional assays with a chemical labeling approach. Instead of performing elaborate experiments to assess protein function, the method uses chemical probes that covalently tag active proteins, substituting complex functional measurement with simpler chemical detection.
3Adaptability or versatility
If family-wide chemical probes are used, then protein family activity can be detected, but precise spatial information is lost due to signal diffusion
Solution Approach 1:
The invention performs preliminary labeling with family-wide chemical probes within intact tissue sections before any extraction or processing. This preliminary action in situ ensures that the spatial distribution of active proteins is captured before diffusion can occur, preserving spatial resolution while maintaining the ability to detect entire protein families.
Solution Approach 2:
The invention segments the detection process to perform family-wide labeling in situ within tissue sections, then extracts the labeled proteins for analysis. This segmentation allows the use of non-specific family-wide probes while preserving spatial information through the in situ labeling step, separating the versatility function from the spatial resolution function.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables quantification of protein activity and function, provides spatial visualization of enzyme activity at sub-cellular and intercellular scales, and increases the dynamic range for measuring low abundance proteins and samples.
Implementation Method 1
the targeting group specifically binds to the active form of the two or more enzymes
Implementation Method 2
incubating the composition under conditions sufficient for the ligation or annealing of the first oligo to the retrieval oligo when the first and retrieval oligos are in close proximity to each other
Implementation Method 3
the first antibody specifically binds to one of the two or more enzymes
Data Source
Figure 1a~1d
Figure 2a~2d
Figure 3a~3h
AI summary
Aspects of the disclosure relate to a method for evaluating two or more target proteins of interest from the same family in a specified functional form, the method comprising: (i) contacting a composition comprising or suspected of comprising the two or more proteins of interest with a molecular construct comprising: a targeting group operatively linked to a retrieval tag; wherein the targeting group specifically binds to the specialized functional form of the two or more target proteins of interest; (ii) contacting the composition with at least two antibody-oligo constructs, wherein at least one of the constructs comprises a first antibody operatively linked to a first oligo and at least a second construct comprises a second antibody operatively linked to a second oligo; wherein the first antibody specifically binds to one of the two or more target proteins of interest and the second antibody specifically binds to the other of the two or more target proteins of interest; (iii) contacting the composition with a second molecular construct comprising a retrieval tag binder operatively linked to a retrieval oligo; (iv) incubating the composition under conditions sufficient for the ligation or annealing of the first oligo to the retrieval oligo when the first and retrieval oligos are in close proximity to each other and ligation or annealing of the second oligo to the retrieval oligo when the second and retrieval oligos are in close proximity to each other; and (v) detecting the ligated or annealed first and retrieval oligo and the ligated or annealed second and retrieval oligo.