Protein Precipitation and Depth Filter Capture for Detergent Removal
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Solution Overview
Problem
Current methods for preparing protein samples for mass spectrometry are hindered by the need to remove detergents like SDS, which are essential for solubilizing hydrophobic proteins but interfere with analysis, and existing techniques such as FASP are time-consuming and require careful handling of spin-filter units.
Innovation Solution
A method involving mild precipitation of proteins with phosphoric acid, followed by capture in a depth filter matrix to prevent aggregation and facilitate detergent removal and in-situ digestion, allowing for efficient processing of small sample amounts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If detergents like SDS are used to solubilize hydrophobic proteins, then protein solubilization is improved, but mass spectrometry analysis is hindered due to detergent interference
Solution Approach 1:
The invention extracts and removes the harmful detergent (SDS) from the protein sample through a series of washing steps using spin-filter units, while retaining the proteins for subsequent mass spectrometry analysis. This resolves the contradiction by separating the solubilization function (achieved during extraction) from the analysis requirement (achieved after detergent removal).
Solution Approach 2:
The method performs preliminary detergent removal and protein precipitation steps before mass spectrometry analysis, ensuring that the sample is prepared in a detergent-free state suitable for MS while having been initially solubilized with detergent to capture hydrophobic proteins.
2Object-affected harmful factors
If conventional FASP method is used for sample preparation, then detergent removal is achieved, but the process becomes time-consuming and requires careful handling
Solution Approach 1:
The sample preparation process is segmented into distinct functional steps (protein precipitation, detergent removal through spin-filters, washing, and digestion) that can be performed in a standardized sequence, improving efficiency and reducing the time required compared to conventional FASP methods.
Solution Approach 2:
The invention employs disposable spin-filter units that are pre-loaded with depth filter material, eliminating the need for repeated handling and cleaning of filter membranes. This disposable approach reduces preparation time and minimizes the risk of contamination or membrane damage.
3Object-affected harmful factors
If spin-filter units are used for detergent removal, then detergent elimination is effective, but reliable performance requires batch-to-batch consistency and careful control
Solution Approach 1:
By using disposable spin-filter units with pre-loaded depth filter material, the invention eliminates variability associated with reusing and cleaning membranes. Each unit is factory-prepared to consistent specifications, ensuring batch-to-batch reliability without requiring careful handling and control during repeated use.
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 rapid, reproducible, and efficient sample preparation that combines detergent removal, protein digestion, and clean-up, improving the processing of hydrophobic proteins and reducing the need for chaotropic agents, thus enhancing proteomic analysis.
Implementation Method 1
exposing said sample to a mild precipitant to cause precipitation of said proteins
Implementation Method 2
bringing said sample into contact with a matrix adapted to capture said precipitated proteins and prevent excessive aggregation of precipitated protein particles
Implementation Method 3
washing the matrix with captured precipitated proteins to remove the surfactant
Data Source
AI summary
The present invention provides a method of preparing a sample comprising one or more proteins of interest, the method comprising: providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium; exposing said sample to a mild precipitant to cause precipitation of said proteins; during or after the precipitation step, bringing said sample into contact with a matrix adapted to capture said precipitated proteins and prevent excessive aggregation of precipitated protein particles; and washing the matrix with captured precipitated proteins to remove the surfactant. A sample preparation device to carry out the same is also provided.