Immobilized Enzyme Digestion for Elevated-Temperature Processing
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Solution Overview
Problem
Existing methods for biomolecule sample processing, such as digestion and affinity ligand purification, face challenges in achieving complete digestion without introducing byproducts, maintaining sample quality, and ensuring reproducibility, especially when conducted at elevated temperatures.
Innovation Solution
The use of immobilized enzymes with minimized secondary interactions on a solid support surface, combined with a tailored digestion buffer and heat source, allows for high-fidelity peptide profiles by reducing non-specific binding and heat-induced modifications, enabling complete digestion in minutes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If enzymes are used in solution for digestion, then the digestion process can be simple, but the enzymes become unstable and form byproducts
Solution Approach 1:
A solid support surface acts as an intermediary carrier for the enzyme, allowing the enzyme to remain stable while facilitating digestion. The enzyme is immobilized on the solid support, preventing direct contact between the enzyme and solution that causes instability, while still enabling substrate access and product release.
2Productivity
If digestion is conducted at elevated temperatures to improve throughput, then workflow efficiency increases, but enzyme denaturation and autolysis increase
Solution Approach 1:
The solid support immobilization changes the thermal stability parameters of the enzyme, allowing it to withstand elevated temperatures without denaturation. This enables digestion to proceed at higher temperatures (improving throughput) while maintaining enzyme integrity (preventing autolysis).
3Reliability
If conventional immobilization methods are used, then enzyme stability improves, but secondary interactions with the surface increase causing non-specific binding
Solution Approach 1:
The solid support surface is designed with differentiated local properties: one region provides immobilization functionality for enzyme attachment, while another region provides anti-fouling properties to minimize non-specific binding. This local quality differentiation allows simultaneous achievement of enzyme stability and reduced harmful interactions.
4Speed
If heat is applied to accelerate digestion, then reaction speed increases, but heat-induced modifications and sample quality degradation occur
Solution Approach 1:
The solid support acts as a heat-distributing intermediary that enables uniform and controlled heating during digestion. This prevents localized overheating and associated heat-induced modifications while maintaining overall reaction speed through efficient thermal transfer.
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
The method achieves >95% sequence coverage with <10% miss-cleavage and <5% heat-induced modification, suitable for biology research and protein therapeutics characterization, while maintaining sample quality and reproducibility.
Implementation Method 1
secondary interactions with the surface of the support (e.g., immobilization solid support) are minimized, greatly reduced, or eliminated
Implementation Method 2
the digestion temperature can be increased. And the drawbacks associated with elevated temperatures can be reduced
Implementation Method 3
immobilized enzymes with minimized secondary interactions on a solid support surface
Implementation Method 4
digestion and affinity ligand purification
Data Source
AI summary
The present disclosure relates to a kit for sample preparation, the kit including a solid support surface with a polymer coating covering the solid support surface, wherein the polymer coating reduces undesired interactions between the sample and the solid support surface, a buffer comprising arginine and methionine, and a vessel for containing the solid support surface and the buffer.