Integrated Protein Extraction and Digestion Workflow
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Solution Overview
Problem
Traditional protein processing methods for body fluids are cumbersome, time-consuming, and prone to sample loss, making it difficult to efficiently extract and analyze low-abundance proteins, which affects the accuracy and throughput of proteome analysis.
Innovation Solution
An integrated method for protein extraction, purification, and digestion is performed in the same centrifuge tube using high molecular polymers, surfactants, solid phase alkylating agents, and proteases, allowing for efficient processing of body fluid proteins without complex operations, and enabling direct analysis by LC-MS.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional multi-step protein processing methods are used, then protein extraction and analysis can be performed, but the process is time-consuming and laborious with inevitable sample loss
Solution Approach 1:
The patent combines multiple separate processing steps (extraction, denaturation, reduction, alkylation, digestion) into a single integrated workflow performed in one tube, eliminating the need for repeated sample transfers between different containers and equipment, thus reducing both time and sample loss while maintaining processing throughput
Solution Approach 2:
The patent segments the processing workflow by performing all steps sequentially in a single container rather than requiring physical transfer between multiple containers, effectively segmenting the process timeline while maintaining spatial continuity of the sample
2Reliability
If traditional multi-step sample transfer methods are used, then protein processing can be completed, but sample loss and pollution occur during transfer
Solution Approach 1:
The patent merges all processing steps into a single-tube workflow, eliminating multiple transfer operations and associated risks of sample loss and contamination, thereby improving sample integrity while reducing operational complexity
Solution Approach 2:
The patent uses a unified buffer system and single-container approach as an intermediary medium that allows all chemical and enzymatic reactions to occur in one continuous environment without exposure to external interfaces that could cause contamination or loss
3Measurement precision
If conventional extraction methods are used, then protein extraction can be performed, but extraction efficiency of low-abundance proteins is insufficient
Solution Approach 1:
The patent employs a systematic optimization of chemical parameters including buffer composition, pH levels, and reagent concentrations to maximize the extraction efficiency of low-abundance proteins while maintaining overall process simplicity and reproducibility
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 method enhances the extraction efficiency of medium and low-abundance proteins, simplifies protein purification, and supports high-throughput clinical proteome analysis with improved accuracy and throughput by maintaining sample continuity and reducing sample transfer-related losses.
Implementation Method 1
A certain proportion of high molecular polymer solution is added to body fluid to extract proteins under low temperature conditions
Implementation Method 2
A certain concentration of surfactant and protein reducing agent are added to rapidly denature the exosome protein under high temperature conditions
Implementation Method 3
the temperature of protein extraction is 80-95° C.
Implementation Method 4
Solid phase alkylating agents capable of rapidly reacting with protein thiol are added to separate proteins from other small molecules
Implementation Method 5
Protease is added and incubates with the microspheres that immobilizes proteins at a certain temperature. The proteinase used may be trypsin, lysine protease, protease V8
Data Source
AI summary
The invention relates to a method for the treatment of body fluid proteins, by which proteins from body fluids such as blood or urine are extracted by adding a certain proportion of high molecular polymer solution under low temperature condition followed by denaturation and reduction by adding a certain concentration of surfactant and tris(2-carboxyethyl) phosphine (TCEP) under a high temperature condition. Subsequently, the iodoacetic acid brushes grafted on silica microspheres called as solid-phase alkylation reagents are added into protein solution, which can react rapidly with the protein sulfhydryl group. After centrifugation, the microspheres are obtained and repeatedly washed with methanol and buffer to remove interferences such as sugars, salts, surfactants, lipids to obtain high-purity proteins, and finally protease is added to digest proteins into peptides. After centrifugation, the peptide products are obtained, and directly analyzed by liquid chromatography-mass spectrometry (LC-MS) system. Compared with the traditional protein pretreatment method, the method has many advantages such as good anti-interference capability, easy operation and short pretreatment time.