Modified Trypsin Polypeptide for Proteomic Sample Purification
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Solution Overview
Problem
Proteomics research and diagnostics face challenges in protein sample preparation due to variations in sample sources, including high salt, DNA, RNA, lipids, and small organic compounds, and the contamination issues with trypsin autolysis peptides during peptide analysis.
Innovation Solution
A modified trypsin polypeptide with biotin moieties and bio-orthogonal coupling pairs is used for protein digestion, allowing for efficient removal of trypsin and purification of peptides through bio-orthogonal click chemistry, using compounds like mTet-CDM and TCO-beads for sample preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If trypsin is used for proteolytic digestion, then protein digestion efficiency is improved, but trypsin autolysis contamination increases
Solution Approach 1:
The invention extracts and removes trypsin from the peptide sample after digestion is complete. This is achieved by adding excess trypsin inhibitor to precipitate trypsin, or by using affinity chromatography to specifically bind and remove trypsin, thereby eliminating the source of autolysis contamination while preserving the digested peptides.
Solution Approach 2:
The invention performs preliminary removal of trypsin immediately after digestion by adding trypsin inhibitor or applying affinity chromatography. This preliminary action prevents trypsin autolysis from occurring during subsequent sample handling and storage, addressing the contamination problem before it can significantly affect the sample.
2Ease of operation
If universal protein sample preparation method is developed, then ease of operation is improved, but manufacturing precision deteriorates
Solution Approach 1:
The invention employs a universal sample preparation protocol that can handle diverse protein samples from various sources (cell lysates, body fluids, tissue extracts). The method uses standardized steps including trypsin digestion with optimized conditions, universal precipitation conditions for trypsin removal, and compatible desalting procedures that work across different sample types, thereby achieving ease of operation.
Solution Approach 2:
The invention optimizes specific parameters within the universal protocol to maintain purification quality. This includes controlling digestion time and temperature, adjusting inhibitor concentration for optimal trypsin precipitation, and fine-tuning buffer compositions to ensure effective peptide recovery while removing contaminants, thus maintaining manufacturing precision across universal applications.
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 effectively purifies peptides by minimizing trypsin autolysis contamination and provides a universal protein/peptide isolation kit for improved proteomic sample preparation, achieving high recovery and purity of proteomic samples.
Implementation Method 1
coupling polypeptides in the sample to a second member of the bio-orthogonal coupling pair linked to a substrate
Implementation Method 2
a compound comprising a member of a bio-orthogonal coupling pair linked to a maleic anhydride moiety
Implementation Method 3
eluting the polypeptides from the substrate in an elution solution having an acidic pH
Data Source
AI summary
Provided herein are methods, reagents, and kits for isolating polypeptides, such as a proteome. Also provided herein is a modified trypsin polypeptide that is resistant to autolysis, and that can be selectively-separated from a biological sample once digestion is complete.