Transgenic Animal Secretome Tagging for Tissue Origin Identification
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Solution Overview
Problem
Current methods fail to accurately identify the tissue origin of proteins in human plasma, which is crucial for understanding health insights and disease biomarkers, as the contribution of specific cell types to the plasma proteome remains elusive.
Innovation Solution
Transgenic non-human animals, such as mice, are engineered to produce tagged polypeptides that are secreted from specific cell types, using proximity-dependent biotin identification (BioID) polypeptides with an ER retention signal, allowing for the identification of biotinylated molecules originating from particular tissues through streptavidin purification and mass spectroscopy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used to analyze plasma proteins, then the analysis can be performed with standard techniques, but the tissue origin of proteins cannot be accurately identified
Solution Approach 1:
The invention segments the plasma proteome analysis by using transgenic animals that express tissue-specific tagged proteins. Each tissue type can be individually tracked through its unique tag (e.g., biotin for endothelial cells), allowing precise identification of protein origins without analyzing the entire complex plasma proteome at once.
Solution Approach 2:
The invention introduces tags (such as biotin, HA-tag, or FLAG-tag) as intermediaries between the proteins and detection methods. These tags serve as mediators that enable specific identification of tissue-origin proteins through affinity purification or immunodetection, bridging the gap between complex plasma samples and accurate tissue origin identification.
2Loss of information
If transgenic animals with tissue-specific tagged protein expression are used, then tissue origin identification is enabled, but the manufacturing and maintenance of the model becomes more complex
Solution Approach 1:
The invention uses universal tagging systems (biotin, HA-tag, FLAG-tag) that can be applied across multiple tissue-specific transgenic models. The same detection and purification methodologies can be used regardless of which tissue is being studied, making the approach universally applicable and reducing the need for tissue-specific custom protocols.
Solution Approach 2:
The invention changes the detectable parameter of proteins from their native sequence (which is difficult to track in plasma) to artificial tags with distinct biochemical properties. This parameter change allows proteins to be identified by their tag rather than their complex native structure, simplifying detection while preserving tissue origin information.
3Measurement precision
If proximity-dependent biotin identification (BioID) is used to tag secreted molecules, then tissue-specific secretomes can be identified, but the purification and analysis process becomes more complex
Solution Approach 1:
The invention extracts tissue-specific proteins from complex plasma samples using affinity purification with streptavidin beads (for biotin tags) or specific antibodies (for HA/FLAG tags). This extraction step isolates the proteins of interest from the overwhelming complexity of the plasma proteome, enabling focused analysis of tissue-specific secretomes without needing to analyze all plasma proteins.
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 the precise identification of tissue-specific secretomes, facilitating the detection of biomarkers for physiological stresses and diseases through non-invasive liquid biopsies by distinguishing biotinylated polypeptides from specific cell types within complex biological samples.
Implementation Method 1
proximity-dependent biotin identification (BioID) polypeptides that include (a) a promiscuous biotin ligase activity such as that derived from Escherichia coli BirA
Implementation Method 2
an endoplasmic reticulum (ER) retention signal... can localize to the lumen of the ER in cells
Implementation Method 3
readily identified as originating from those cells using streptavidin or avidin purification followed by amino acid analysis
Implementation Method 4
amino acid analysis (e.g., mass spectroscopy)
Data Source
AI summary
This document relates to methods and materials involved in the deconvolution of serum. For example, transgenic non-human animals (e g , transgenic mice) that secrete tagged (e.g., biotinylated) molecules from a particular tissue are provided.