# Corundum Particles as Trypsin Carrier for Efficient Protein Digestion

## TL;DR

Researchers developed a low-cost, reusable corundum-based carrier for trypsin, improving protein digestion efficiency and stability.

## Contribution

A novel, cost-effective method for immobilizing trypsin on corundum particles is introduced, enhancing digestion performance and reusability.

## Key findings

- Corundum-immobilized trypsin showed improved stability in 1 M guanidinium hydrochloride and retained >80% activity across reuse cycles.
- Digestion of NISTmAb and Herceptin achieved comparable or better peptide yields and sequence coverage than free trypsin.
- The immobilized enzyme exhibited a higher temperature optimum (60 °C) and preserved functionality for weeks at 4 °C.

## Abstract

Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80% across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved >60% sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows.