Target Protein Extraction Using Ammonium Salt Precipitation
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Solution Overview
Problem
Current methods for extracting target proteins from biological samples, such as serum or plasma, are inefficient and often result in unreliable measurement stability due to the presence of contaminating proteins.
Innovation Solution
The method involves adding a high concentration of salt and/or urea, along with a water-soluble organic solvent, to the biological sample to efficiently extract the target protein, thereby enhancing recovery rates and measurement precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If conventional deproteinization methods (acid/organic solvent addition) are used to remove contaminating proteins, then high molecular weight proteins can be removed, but the method is often ineffective for various target proteins and unexpected chemical reactions may occur
Solution Approach 1:
The patent changes the chemical parameters by using ammonium salts (ammonium formate, ammonium acetate, ammonium hydrogen carbonate, ammonium sulfate) instead of traditional acids or organic solvents. This parameter change allows for selective precipitation of high molecular weight proteins while maintaining the solubility and stability of target proteins, thereby resolving the contradiction between removing contaminants and preserving target protein integrity
Solution Approach 2:
The patent introduces ammonium salts as an intermediary substance that mediates the deproteinization process. The ammonium salts act as a selective precipitating agent that interacts with high molecular weight proteins to form insoluble complexes, while leaving target proteins in solution. This intermediary approach avoids direct harsh treatment of target proteins, resolving the reliability issue
2Object-affected harmful factors
If physical removal methods (ultrafiltration, dialysis, ultracentrifugation) are used to separate target proteins, then mild conditions are maintained with little side reaction, but considerable effort is required for preliminary experiments and membrane selection
Solution Approach 1:
The patent replaces mechanical separation methods (ultrafiltration, dialysis, ultracentrifugation) with a chemical precipitation method using ammonium salts. This substitution eliminates the need for complex mechanical systems and preliminary experiments to select membranes or optimize centrifugation conditions. The chemical method provides a more straightforward protocol that can be applied generally without extensive preliminary work
Solution Approach 2:
The patent changes the approach from physical separation parameters (membrane cutoff, centrifugation speed, dialysis time) to chemical parameters (ammonium salt concentration, pH). This parameter change simplifies the process by using readily adjustable chemical conditions instead of complex mechanical parameters that require preliminary optimization
3Measurement precision
If high performance liquid chromatography and mass spectrometry are used for trace protein analysis, then high measurement precision is achieved, but the methods require highly specific conditions and are not easily performed in clinical sites
Solution Approach 1:
The patent performs preliminary deproteinization treatment using ammonium salts before the actual analysis. This preliminary action removes contaminating proteins and prepares the sample in advance, thereby simplifying the subsequent analysis steps. The pre-treatment enables the use of simpler, more accessible instrumentation in clinical settings while maintaining measurement precision
Solution Approach 2:
The patent extracts and removes high molecular weight contaminating proteins using ammonium salt precipitation before the target protein analysis. This extraction step cleans up the sample matrix, thereby reducing the complexity requirements for subsequent measurement instruments. The extracted contaminants are separated from the target proteins, enabling the use of more accessible analytical equipment in clinical environments
4Adaptability or versatility
If manual measurement methods are used for protein analysis, then flexibility is maintained, but troublesomeness and reproducibility are compromised
Solution Approach 1:
The patent standardizes the deproteinization process by using defined ammonium salt concentrations and conditions, thereby transforming manual measurement into a more standardized protocol. This parameter standardization reduces variability between measurements and operators, thereby improving reproducibility while maintaining flexibility through the simplicity of the ammonium salt method
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 allows for the efficient extraction of target proteins with high recovery rates, leading to more precise and sensitive analyses, and enables faster handling of large specimen amounts, particularly in clinical settings.
Implementation Method 1
a method in which insolubilization caused by protein denaturation is utilized... the higher-order structure of a protein is broken to denature and precipitate the protein
Implementation Method 2
the higher-order structure of a protein is broken to denature and precipitate the protein
Implementation Method 3
a water-soluble organic solvent is used... the target protein is one or more selected from a group consisting of proteins that can be dissolved in a water-soluble organic solvent
Data Source
AI summary
An object of the present invention is to provide a method of efficiently extracting a target protein contained in a biological sample such as serum or plasma, thereby enabling highly precise analysis. In the present invention, there is provided the method in which a protein is removed from a sample derived from a living body to extract the target protein, and a salt and/or urea at a high concentration, and a water-soluble organic solvent are used.