Single Molecule Proteomics via Polypeptide Unfolding and Residue Tagging
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Solution Overview
Problem
Current methods for analyzing protein samples, especially complex mixtures like the human proteome, face challenges in identifying medium to low abundance proteins due to high dynamic range and complexity, requiring effective fractionation, enrichment, and detection techniques that can handle single molecule analysis.
Innovation Solution
The method involves unfolding proteins into polypeptides, labeling specific residues, and analyzing their linear length and pattern using techniques like nanopore translocation and imaging to determine the location, distance, and order of residues, which are then compared to a database for protein identification, incorporating techniques such as DNA PAINT for super-resolution imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional protein analysis methods (immunoassays, microarrays, mass spectrometry) are used, then high abundance proteins can be detected, but medium to low abundance proteins cannot be effectively identified due to high dynamic range and complexity
Solution Approach 1:
The protein analysis process is segmented into distinct stages: fractionation to separate protein groups, enrichment to concentrate target proteins, and single-molecule detection to identify individual proteins. This segmentation allows each stage to be optimized independently, improving overall detection sensitivity for low abundance proteins while managing complexity through modular processing
Solution Approach 2:
Fractionation and enrichment procedures are performed as preliminary actions before the actual protein detection. By pre-processing the complex protein mixture to reduce dynamic range and concentrate target proteins, the subsequent detection step can focus on identifying medium to low abundance proteins with higher precision without being overwhelmed by the full complexity of the original mixture
2Measurement precision
If proteins are analyzed in their native folded state, then functional information is preserved, but structural complexity prevents accurate linear length and residue pattern analysis
Solution Approach 1:
The physical state parameter of proteins is changed from folded to unfolded through denaturation. This parameter change transforms the three-dimensional folded structure into a linear polypeptide chain, enabling accurate measurement of linear length and residue positions while preserving the amino acid sequence information necessary for protein identification and functional analysis
3Measurement precision
If single molecule detection methods are implemented, then detection sensitivity for low abundance proteins is improved, but measurement precision and signal detection become more challenging
Solution Approach 1:
Detectable labels (such as fluorophores or other signal-generating moieties) are used as intermediaries to attach to specific residues on the polypeptide chain. These labels amplify the signal from individual molecules, making single molecule detection feasible by converting weak intrinsic signals into detectable signals that can be measured with sufficient precision
Solution Approach 2:
Detectable labels that produce optical signals (such as fluorescence) are attached to specific residues. The presence, position, and pattern of these labeled residues create a characteristic signal pattern or 'fingerprint' that can be detected and used to identify the protein, transforming the invisible single molecule into a detectable signal through optical property changes
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 the identification of proteins in complex mixtures by reducing the dynamic range and enhancing the detection of low abundance proteins, allowing for precise fingerprinting and identification of proteins in biological samples with high accuracy.
Implementation Method 1
the length of the polypeptide is analysed by passing it through a nanopore or nanogap and the pattern or location of distinct residues is determined by recording the length of time between signals
Implementation Method 2
recording the length of time between signals
Implementation Method 3
incorporating techniques such as DNA PAINT for super-resolution imaging
Implementation Method 4
The distinct residues can be determined by a number of means including labeling them with detectable labels
Data Source
AI summary
This disclosure comprises devices and methods for determining the identity of individual protein molecules in a complex mixture by unfolding the protein into a polypeptide, tagging selected residues on the polypeptide with selected oligonucleotide sequence tags that recognize selected residues on said polypeptide, and then detecting the oligonucleotide sequence tags.