Protein Quantification via Cleaved Peptide Barcodes
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Solution Overview
Problem
Current methods for proteomics are hindered by the scale, dynamic range, and inability to amplify proteins, making it difficult to fully capture the complex and dynamic states of cells.
Innovation Solution
A method involving the use of fusion polypeptides where a protein of interest is fused to a peptide barcode. The peptide barcode is then cleaved and sequenced to identify the protein of interest, enabling protein identification and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional proteomics methods are used, then protein analysis can be performed, but the scale and dynamic range are limited and proteins cannot be amplified
Solution Approach 1:
The patent uses peptide barcodes as informational copies that can be amplified independently of the protein. Instead of directly analyzing the protein, the method creates a separable peptide barcode copy that carries identification information, allowing amplification of the barcode signal while the protein remains stable and functional. This resolves the contradiction by enabling quantity amplification through barcode copying rather than protein amplification.
Solution Approach 2:
The patent segments the protein analysis function into two independent parts: the protein itself (which maintains structural and functional integrity) and the peptide barcode (which can be separated, amplified, and sequenced). This segmentation allows the barcode to be amplified independently to improve detection sensitivity while the protein maintains its original properties, resolving the scale and amplification limitations.
2Measurement precision
If fusion polypeptides with peptide barcodes are used, then protein identification and quantification are enabled, but the method complexity increases
Solution Approach 1:
The peptide barcode acts as an intermediary element between the protein and the sequencing system. Instead of directly sequencing the protein (which would be complex and difficult), the barcode serves as a mediator that simplifies the identification process. The barcode can be easily separated, amplified, and sequenced, providing a simpler pathway to protein identification while maintaining precise measurement capabilities.
Solution Approach 2:
The patent creates a simplified copy (peptide barcode) that carries the identification information needed for protein analysis. This copy is much simpler to handle, amplify, and sequence than the protein itself, thereby reducing method complexity while maintaining measurement precision through the barcode's unique sequence information.
3Ease of operation
If the peptide barcode is cleaved from the fusion polypeptide, then the barcode can be sequenced to identify the protein, but the protein must be fused to the barcode initially
Solution Approach 1:
The patent performs preliminary action by fusing the peptide barcode to the protein before analysis. This pre-attachment ensures that the barcode is positioned correctly and can be easily separated and sequenced afterward. The fusion step is performed once during protein preparation, and subsequent steps (separation, amplification, sequencing) become simpler and more straightforward, improving ease of operation while the fusion requirement is established in advance.
Solution Approach 2:
The patent extracts the peptide barcode from the fusion polypeptide through cleavage, separating it from the protein. This extraction allows the barcode to be independently handled, amplified, and sequenced without the complexity of analyzing the entire fusion protein. The cleavage step enables the barcode to be taken out as a separate, analyzable entity, improving ease of operation for sequencing while the initial fusion requirement is resolved by the extraction process.
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 method allows for the efficient identification and quantification of proteins, overcoming the limitations of existing proteomics approaches by providing a scalable and dynamic means to analyze protein states in cells.
Implementation Method 1
contacting the fusion polypeptide with a cleaving agent, wherein the cleaving agent cleaves the peptide barcode from the fusion polypeptide comprising the protein of interest
Implementation Method 2
sequencing the peptide barcode to identify the protein of interest
Data Source
AI summary
The present disclosure provides methods and compositions for protein quantification using peptide barcodes, sample preparation for protein quantification, protein quantification using error-resistant peptide barcodes, methods to manipulate barcoded proteins, methods to determine the binding affinities of many protein variants in a mixture of proteins by single molecule measurements.