Dithioester Peptide Sequencing Reagents for Spatial Detection
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Solution Overview
Problem
Current protein sequencing methods, such as mass spectrometry and Edman degradation, lack single molecule sensitivity and spatial information, while immunohistochemistry provides limited scalability and sequence information, making it difficult to quantify and identify low copy-number proteins effectively.
Innovation Solution
A sequencing reagent with dithioester or thiocarbamoyl reactive groups forms covalent bonds with N-terminal amino acids, allowing for the tethering and detection of polypeptides, which can be sequenced using a method involving cleavable or non-cleavable linkers, polymerization, and nanopore translocation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used for protein identification, then protein quantification is enabled, but single molecule sensitivity and spatial information are lacking
Solution Approach 1:
The patent introduces a DNA oligonucleotide mediator that bridges the protein of interest and the solid substrate. The oligonucleotide contains a capture sequence that binds to the protein and a complementary sequence that hybridizes to substrate-bound primers, enabling single molecule detection while preserving spatial information through the DNA-mediated connection
Solution Approach 2:
The patent replaces traditional mass spectrometry detection with a nanopore-based electrical detection system. Protein sequences are transcribed to DNA, which then translocates through a nanopore for detection, substituting the mechanical mass/charge ratio measurement with an electrical signal-based system that provides both sensitivity and spatial information
2Measurement precision
If Edman degradation is used for protein sequencing, then sequential amino acid identification is achieved, but throughput is low and harsh conditions are required
Solution Approach 1:
The patent replaces the chemical Edman degradation process with a biological transcription-translation-nanopore detection system. Instead of iterative chemical reactions requiring harsh conditions, the system uses enzymatic transcription to DNA followed by single-pass nanopore sequencing, achieving high throughput under physiological conditions
Solution Approach 2:
The patent fundamentally changes the detection parameters from chemical mass spectrometry or iterative chemical degradation to electrical signal detection through a nanopore. This parameter change enables parallel processing of multiple molecules simultaneously, dramatically increasing throughput while maintaining accuracy
3Loss of information
If immunohistochemistry is used for protein visualization, then spatial localization is provided, but sequence information and scalability are limited
Solution Approach 1:
The patent creates a universal platform where a single oligonucleotide-tagged protein preparation can be used across multiple proteins in the proteome. The DNA-mediated capture and nanopore detection system provides both spatial information through substrate binding and sequence information through transcription, enabling proteome-wide analysis with a unified methodology
Solution Approach 2:
The patent uses an oligonucleotide mediator that serves dual functions: maintaining spatial information through substrate-bound primer hybridization and enabling sequence detection through transcription. This DNA intermediary bridges the gap between spatial visualization and sequence identification, providing both types of information simultaneously
4Quantity of substance
If current methods are used for low copy-number protein detection, then some proteins are identified, but about 10% of mammalian protein expression remains undetected
Solution Approach 1:
The patent replaces mass spectrometry detection with single-molecule nanopore detection, achieving attomole and subattomole sensitivity. The electrical signal detection through the nanopore allows direct observation of individual protein molecules, dramatically improving detection sensitivity for low copy-number proteins
Solution Approach 2:
The patent introduces an oligonucleotide capture mediator that concentrates and tethers target proteins to the solid substrate, enabling single molecule detection. This mediator amplifies the detection signal by providing a stable platform for nanopore translocation, improving sensitivity for low abundance 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 high-throughput, single molecule sequencing of proteins with spatial information, overcoming the limitations of existing methods by providing sensitive and scalable protein identification.
Implementation Method 1
A comprises a (e.g., first) reactive group configured to form a covalent bond with an N-terminal amino acid of a polypeptide
Implementation Method 2
nanopore translocation
Data Source
AI summary
The present disclosure provides reagents and methods useful for single-molecule sequencing of proteins through use of a unique sequencing reagent. The reagents and methods described herein provide for high-throughput single-molecule peptide and protein sequencing in mild conditions allowing for high resolution investigation of the complex biological systems.