In Vivo Proteomic Mapping via Promiscuous Tagging Enzymes
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Solution Overview
Problem
Current proteomics methods are unable to provide a comprehensive assessment of the proteome of living cells or substructures within them, as they require cell lysis and cannot be performed on living cells, leading to spatial and dynamic information loss and issues with false positives and negatives in purification processes.
Innovation Solution
The development of strategies and methods for in vivo proteomics that allow for the labeling of proteins within living cells using promiscuous tagging enzymes, which can be genetically targeted to specific subcellular regions, enabling proteomic mapping without the need for sample purification, using enzymes like peroxidases to catalyze reactions that covalently label nearby proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional proteomics methods are used with cell lysis and purification, then protein identification is possible, but spatial and dynamic information is lost and false positives/negatives occur
Solution Approach 1:
The patent applies preliminary action by introducing a promiscuous tagging enzyme into living cells before proteome analysis. The enzyme is genetically targeted to specific subcellular regions and catalyzes covalent labeling of nearby endogenous proteins with a tagging substrate. This preliminary labeling action occurs in vivo, preserving spatial information, and allows subsequent proteomic analysis without cell lysis, thereby maintaining dynamic information and avoiding purification-related false positives/negatives.
2Adaptability or versatility
If purification methods are used to isolate subcellular structures, then specific compartments can be analyzed, but numerous false positives and false negatives occur and many structures cannot be purified
Solution Approach 1:
The patent uses a promiscuous tagging enzyme as an intermediary to bridge the gap between genetic targeting capability and proteome analysis. The enzyme is genetically targeted to any subcellular region of interest and serves as a mediator that catalyzes covalent labeling of nearby endogenous proteins. This intermediary approach eliminates the need for physical purification of subcellular structures, thereby achieving universal accessibility to all subcellular compartments while eliminating purification-related false positives and false negatives.
3Productivity
If mass spectrometry is used for unbiased protein identification, then nearly all proteins can be identified, but the method cannot be performed on living cells
Solution Approach 1:
The patent applies preliminary action by performing covalent labeling of proteins in vivo before any analysis step. The promiscuous tagging enzyme, genetically targeted to specific subcellular regions, catalyzes the attachment of tagging substrates to nearby endogenous proteins while cells remain alive. This preliminary labeling preserves the living state of cells during the labeling process, enabling subsequent proteomic analysis with high throughput while maintaining living cell compatibility.
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
This approach allows for accurate and high-throughput analysis of proteomes in living cells, enabling the creation of proteome maps for different cells, subcellular compartments, and organisms under various conditions, including those exposed to therapeutic agents, and provides detailed information on protein interactions and compositions.
Implementation Method 1
contacting a living cell with a tagging enzyme under conditions suitable for the tagging enzyme to catalyze a reaction with a tagging substrate resulting in the tagging of proteins within the vicinity of the tagging enzyme
Data Source
AI summary
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.