Protein Conformation Detection via Limited Proteolysis and Mass Spectrometry
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Solution Overview
Problem
Current methods for determining protein conformation and conformational changes in their native biological context are limited, particularly in complex biological matrices, and lack the ability to detect conformational changes in a multiplexed manner.
Innovation Solution
A novel MS-based approach that focuses on identifying unique structural/conformational states of proteins by performing limited proteolysis under non-denaturing conditions, followed by filtration to remove large peptides and proteins, and then analyzing the remaining peptides using LC-MS and DIA mass spectrometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional biophysical techniques (NMR, X-ray crystallography, fluorescence spectroscopy) are used to monitor protein conformational changes, then measurement precision is improved, but device complexity and ease of operation worsen due to requirement for purified proteins in vitro and inability to handle complex biological backgrounds
Solution Approach 1:
The patent replaces complex biophysical measurement systems (NMR, X-ray crystallography, fluorescence spectroscopy) with a mass spectrometry-based proteolytic approach. Instead of using sophisticated instrumental systems that require purified proteins, the invention uses enzymatic proteolysis combined with MS to detect conformational changes in complex biological matrices, thereby reducing system complexity while maintaining measurement capability
Solution Approach 2:
The patent introduces proteolytic enzymes as intermediaries to detect protein conformational changes. The enzymes' proteolytic activity is influenced by protein structure and conformation, providing a measurable signal that reflects conformational states without requiring direct observation of the protein itself through complex biophysical instruments
2Adaptability or versatility
If FRET-based techniques are used to monitor conformational changes in native cellular environment, then adaptability is improved, but device complexity and ease of operation worsen due to requirement for fluorescent probe introduction and inability to scale to large samples
Solution Approach 1:
The patent employs proteins themselves as the probe through their native conformational states. Instead of introducing external fluorescent probes that complicate the system, the invention uses the proteins' intrinsic structural properties and their differential proteolytic susceptibility to provide the measurement signal, thereby simplifying operation while maintaining native environment monitoring capability
Solution Approach 2:
The patent extracts conformational information through proteolytic fragmentation rather than through fluorescent labeling. By using proteases to generate peptide fragments whose patterns reflect protein conformation, the invention removes the need for complex probe introduction and enables scaling to large sample sizes without operational complications
3Ease of operation
If limited proteolysis combined with mass spectrometry is used to detect conformational changes in complex biological matrices, then ease of operation is improved, but measurement precision worsens due to complexity of biological backgrounds and difficulty in detecting conformational changes
Solution Approach 1:
The patent segments proteins into peptide fragments through controlled proteolysis. This segmentation creates measurable differences in fragment patterns that reflect conformational changes, even in complex biological matrices. The segmented peptide fragments provide distinct spectral signatures that can be precisely measured by mass spectrometry, thereby improving detection accuracy while maintaining operational simplicity
Solution Approach 2:
The patent changes the physical-chemical parameters of protein detection by using proteolytic fragment mass-to-charge ratios instead of direct protein conformational parameters. This parameter transformation enables precise measurement of conformational changes through mass spectrometry of peptide fragments, overcoming the difficulty of detecting conformational changes in complex biological backgrounds while keeping the method operationally simple
4Productivity
If conventional proteomic techniques are used to probe cellular protein concentration, then productivity is improved, but loss of information worsens due to inability to detect conformational changes
Solution Approach 1:
The patent makes the proteomic analysis system multi-functional by enabling it to detect both protein concentration changes and conformational changes through a single integrated approach. By combining limited proteolysis with mass spectrometry, the system can simultaneously quantify protein abundance and detect conformational states, thereby preventing information loss while maintaining high productivity through multiplexed analysis
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 detection of protein structural changes with high sensitivity, coverage, and throughput, providing valuable information on protein structure and function, and potentially serving as biomarkers for disease detection.
Implementation Method 1
Limited proteolysis of the complex (e.g. cell extract) mixture under a condition in which the at least one protein is in the original conformational state to be detected
Implementation Method 2
Removal of large peptides and proteins or other biomolecules from said first fragment sample
Implementation Method 3
analytical analysis of the enriched fragment sample for the determination of fragments characteristic of having been the result of the limited proteolysis
Data Source
AI summary
Method for the detection of a conformational state of a protein being in a complex mixture of further proteins and other biomolecules, wherein the protein has been subjected to a condition inducing a structural change, including: limited proteolysis of the extract mixture under a condition in which the protein is in the original conformational state to be detected leading to a first fragment sample; directly followed by (2) removal of large peptides and proteins or other biomolecules from said first fragment sample to form an enriched fragment sample; (3) analytical analysis of the enriched fragment sample for the determination of fragments characteristic of having been the result of the limited proteolysis of (1) as well as remaining after the removal (2) for the determination of the conformational state of said at least one protein.