Authors: Siene Swinnen
Source: Lirias (KU Leuven) (article, 2026-02-06)
Field: Chemistry — Inorganic Chemistry

Ontwikkeling van een nieuw protocol voor metaal-organische roosters (MOFs)als artificiële enzymen in proteomics-toepassingen The development of artificial proteases responds to an increasing demand for robust, controllable, and scalable alternatives to enzyme-based methodologies. Conventional proteomics workflows rely heavily on biological proteases such as trypsin, which, despite their high specificity, suffer from limitations including narrow operational windows and susceptibility to denaturation. Simultaneously, advances in materials chemistry have positioned MOFs as attractive catalysts with enzyme-like functions. The convergence of proteomics and MOF chemistry represents a strategic opportunity to rethink protein processing from a materials-driven perspective beyond the constraints of natural enzymes. Zirconium-based metal-organic frameworks have emerged as robust platforms for bioinspired catalysis, particularly in proteomics, where controlled protein cleavage is crucial for the sample preparation workflow. Built from inorganic Zr-oxo clusters linked by organic ligands, these frameworks combine structural tunability with excellent chemical stability and reactivity, offering advantages over natural proteases, such as long-term stability, recyclability, and potentially alternative cleavage selectivity. Despite these promising features, several challenges must be addressed to enable the mitigation of MOFs into high-throughput proteomics platforms, as discussed in Chapter 1. This research addresses alternative methods to fine tune MOF reactivity, investigates the framework-protein interactions, and explores the mechanism of selectivity directing MOF-mediated protein cleavage. A central finding of this dissertation is that substrate adsorption dynamics strongly influence the apparent efficiency of hydrolysis of peptides and proteins. In Chapter 2, we characterized the reactivity of UiO-66 MOFs differing in metal node composition by comparing UiO-66 built with [Zr₆O₈] or [Zr₁₂O₂₂] clusters nodes. The latter achieved a 10,000-fold rate enhancement under mild reaction conditions, however, strong binding of the reaction product glycine decelerates overall conversion. These findings highlight the delicate balance between reactivity and product release, and demonstrates how cluster nuclearity profoundly affects the hydrolytic efficiency. Chapter 3 aims to design a potential solution for the strong adsorption of reaction products onto the framework of MOFs. Various elution conditions were tested after hydrolysis reactions promoted by UiO-66 to allow complete peptide fragment recovery. These results reveal that the true reactivity of UiO-66 had been underestimated in earlier studies, as incomplete recovery masked its performance. Since the adsorption and desorption dynamics is a determining factor for the eventual reactivity of MOF nanozymes, we set out to explore in Chapter 4 the protein-MOF interactions and adsorption kinetics through in situ infrared spectroscopy. The encapsulation efficiency and secondary structure retention strongly depend on the pH and ionic strength, indicating the importance of electrostatic interactions, rather than solely adsorption based on the hydrophobic effect. Furthermore, in Chapter 5 electrostatic interactions were again found important in the recruiting step of the hydrolysis reaction mechanism. Electrophoresis, mass spectrometry and molecular modeling were combined to unravel the structural and mechanistic basis behind the selective protein hydrolysis of MOFs. UiO-66's found selectivity contrasts starkly with discrete Zr clusters, which favor cleavage near aspartic acid. UiO-66 preferentially hydrolyzed peptide bonds of cytochrome c adjacent to glycine, threonine, and lysine residues, especially when these residues occurred in close proximity. This unique reactivity demonstrates not only the metal influences the selectivity of MOFs, but also the interactions of their framework. Generally, this work demonstrates that Zr-MOFs can serve as selective, stable protease mimics. By addressing adsorption, product release, and selectivity, this research advances the mechanistic understanding and practical solutions for integrating MOFs into proteomic workflows. Beyond proteomics, these findings clarify fundamental biomolecule-framework interactions, guiding the design of next-generation nanozymes that bridge heterogeneous catalysis and biotechnology. Proteomics ; Scalability ; Proteases ; Biopharmaceutical ; Workflow ; Posttranslational modification ; Cleavage (geology) Metal-Organic Frameworks: Synthesis and Applications ; Advanced Nanomaterials in Catalysis ; Metal complexes synthesis and properties