Corundum Particles as Trypsin Carrier for Efficient Protein Digestion.

Area of Science:

• Biochemistry
• Proteomics
• Materials Science

Background:

• Reusable enzyme carriers are crucial for efficient proteomic workflows.
• Existing supports are often expensive or lack durability.
• Need for robust and cost-effective enzyme immobilization methods.

Purpose of the Study:

• To covalently immobilize recombinant trypsin onto micrometer-sized corundum particles.
• To evaluate the performance of immobilized trypsin in protein digestion and antibody analysis.
• To assess the cost-effectiveness and robustness of the developed enzyme carrier.

Main Methods:

• Corundum particle surface modification via cleaning, silanization, and glutaraldehyde activation.
• Covalent attachment of recombinant trypsin and reduction of imines.
• Enzyme loading quantification using Aromatic Amino Acid Analysis (AAAA) and non-specific adsorption blocking.

Main Results:

• Enzyme loading achieved was approximately 1 µg/mg.
• Immobilized trypsin exhibited enhanced thermal stability and stability in 1 M guanidinium hydrochloride.
• Activity remained high (>80%) over multiple reuse cycles and preserved functionality during storage.
• Comparable peptide yields and sequence coverage to soluble trypsin for NISTmAb digestion, outperforming it at elevated temperatures.
• Successful antibody identification and >60% sequence coverage for Herceptin digests using MALDI-TOF MS.

Conclusions:

• Corundum-immobilized trypsin offers a cost-effective and robust alternative for proteomic applications.
• The developed carrier demonstrates excellent analytical performance in protein digestion and antibody analysis.
• This method presents an attractive option for both research and routine proteomic workflows.