Protein Nanopore Characterization for Single-Molecule PTM Mapping
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Solution Overview
Problem
Current methods for characterizing peptides, polypeptides, and proteins, such as mass spectrometry and Edman degradation, are unsuitable for single-molecule analysis and struggle with accurately mapping post-translational modifications, especially when they are present in only a fraction of the sample, and methods using nanopores face challenges like irregular movement and experimental complexity.
Innovation Solution
The method involves using an engineered protein nanopore with a solvent-accessible channel and non-native charged moieties, driven by electroosmotic force to translocate peptides and proteins in a linearized state, allowing for measurements that characterize their properties, including post-translational modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used to characterize peptides and proteins, then bulk information about the sample can be obtained, but it is not a single molecule technique and cannot accurately map modifications present in only a fraction of peptides
Solution Approach 1:
The patent replaces traditional mass spectrometry mechanical systems with a nanopore-based electrical measurement system. A single peptide or protein molecule is translocated through a nanopore under an applied electric field, and modifications are detected through changes in ionic current as the molecule passes through the pore, enabling single-molecule characterization without the complexity of mass spectrometry instrumentation.
Solution Approach 2:
The patent changes the detection parameter from mass-to-charge ratio (used in mass spectrometry) to ionic current blockage characteristics. By monitoring how a single peptide or protein molecule affects ion flow through the nanopore, the system can detect post-translational modifications with high precision at the single-molecule level, resolving the contradiction between measurement precision and device complexity.
2Ease of operation
If electrophoretic force is used to drive a charged polymer through a nanopore, then the peptide can be threaded through the nanopore, but the movement becomes irregular after the leader sequence exits the pore
Solution Approach 1:
The patent introduces an intermediary mechanism - electroosmotic flow generated by charged moieties within the nanopore channel - to mediate the translocation process. This electroosmotic pump creates a controlled flow that drives the peptide through the pore in a regular, predictable manner, replacing the irregular electrophoretic movement and improving translocation reliability while maintaining ease of operation.
3Ease of operation
If chemical attachment of a leader sequence is performed to enable nanopore threading, then the peptide can be translocated through the nanopore, but the structure or properties of the underlying native peptide may be altered
Solution Approach 1:
The patent extracts and eliminates the need for chemical modification of the peptide. By designing the nanopore with internal charged moieties that generate electroosmotic flow, the system achieves translocation capability without requiring attachment of leader sequences, thereby preserving the native structure and composition of the peptide or protein being analyzed.
4Ease of operation
If processive enzymes such as unfoldases are used to ratchet a peptide through a nanopore, then translocation can be achieved, but experimental conditions may not be compatible with retention of enzymatic activity
Solution Approach 1:
The patent replaces the biological mechanical system (processive enzymes like unfoldases) with a physical electroosmotic pump. The charged moieties in the nanopore generate electroosmotic flow that drives translocation without requiring enzymatic activity, allowing the use of any experimental conditions compatible with peptide stability while maintaining ease of operation and translocation capability.
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 accurate, single-molecule characterization of peptides and proteins, including mapping of widely separated post-translational modifications without chemical alteration, suitable for commercial nanopore devices and potentially single-cell proteomics.
Implementation Method 1
under conditions such that an electroosmotic force across the nanopore causes the peptide, polypeptide or protein to translocate through the nanopore in a linearised state
Data Source
AI summary
Provided herein are methods of characterising a peptide, polypeptide or protein and of characterising one or more proteoforms of a peptide, polypeptide or protein, using nanopores. Also provided herein are associated systems.