Polypeptide Sequencing via Terminal Amino Acid Extraction
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Solution Overview
Problem
Current methods for high-throughput polypeptide sequencing face challenges in achieving specific and stable recognition of terminal amino acid residues, particularly due to variability in binding affinity influenced by neighboring amino acids, and lack efficiency and reversibility in processing multiple binding agents and polypeptides simultaneously.
Innovation Solution
The method involves using a coupler to bind to the terminal amino acid of a polypeptide, attaching the coupler-polypeptide complex to a solid support, cleaving the peptide bond to isolate the terminal amino acid, and using a binding agent with a coding tag to identify the amino acid, allowing for repeat cycles and parallel processing of multiple polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If binding agents are used to recognize terminal amino acid residues, then polypeptide sequencing can be performed, but binding affinity and specificity vary depending on neighboring amino acids (P2, P3 residues)
Solution Approach 1:
The patent extracts and isolates the terminal amino acid residue from its native polypeptide context by cleaving the peptide bond between P1 and P2 residues. This extraction removes the influence of neighboring amino acids (P2, P3 residues) that cause variability in binding affinity, allowing binding agents to recognize the terminal amino acid with consistent and reliable affinity regardless of the original polypeptide sequence.
Solution Approach 2:
The patent segments the polypeptide chain by cleaving the peptide bond adjacent to the terminal amino acid residue. This segmentation separates the target amino acid (P1) from its neighboring residues (P2, P3), creating an isolated amino acid-coupler complex that can be recognized by binding agents with uniform affinity, thereby improving measurement precision and reliability.
2Productivity
If multiple binding agents and polypeptides are processed simultaneously, then high-throughput sequencing is achieved, but efficiency and reversibility of processing are insufficient
Solution Approach 1:
The patent employs a universal coupler molecule that can simultaneously bind to multiple different terminal amino acid residues and serve as a common attachment point for various binding agents. This universal coupler enables parallel processing of multiple polypeptides with different binding agents in the same reaction system, achieving high-throughput sequencing while maintaining processing efficiency and allowing reversible binding for repeated cycles.
3Reliability
If crosslinking reagents are used to stabilize binding agents, then binding stability is improved, but processing speed and reversibility are reduced
Solution Approach 1:
The patent changes the binding parameter from irreversible crosslinking to reversible non-covalent binding. The coupler-amino acid complex forms stable but reversible interactions with binding agents, allowing the system to maintain binding stability for accurate identification while enabling rapid dissociation and reuse of binding agents for subsequent sequencing cycles, thereby improving both speed and reversibility.
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 enables accurate, high-throughput, and parallel identification of terminal amino acid residues with improved specificity and stability, independent of neighboring amino acids, facilitating efficient polypeptide sequencing.
Implementation Method 1
contacting the terminal amino acid residue of the polypeptide with a coupler, followed by attaching the coupler-polypeptide complex to a solid support
Implementation Method 2
cleaving the peptide bond between the terminal amino acid and the solid support
Data Source
AI summary
The present disclosure relates to methods and kits for performing an identification of a terminal amino acid residue of the polypeptide, or performing a polypeptide sequencing. The methods include a step of contacting the terminal amino acid residue of the polypeptide with a coupler, followed by attaching the coupler-polypeptide complex to the solid support and cleaving the coupler-polypeptide complex from the polypeptide, thereby isolating the terminal amino acid residue of the polypeptide from the remaining amino acid residues of the polypeptide in complex with the coupler, thereby enabling efficient identification of the terminal amino acid residue via recognition by binding agents capable of binding to the coupler-amino acid complex. In some embodiments, the coupler and the polypeptide are both associated with stabilizing components, and after binding of the coupler to the terminal amino acid of the polypeptide, tethering complex is formed between the stabilizing components releasably attached to the solid support.