Using High‐Throughput Screening to Identify Crosslinking Peptides That Control Cell‐Mediated Matrix Degradation

Cells modify the extracellular matrix by expressing proteases that degrade matrix proteins, enabling cell migration within tissues. This process is mimicked in hydrogels through protease-degradable peptide crosslinks. However, cleaving hydrogel crosslinks reduces local matrix mechanical properties, and most crosslinking peptides, including the widely used GPQGIWGQ "PanMMP" sequence, often lead to bulk hydrogel degradation. Membrane-type proteases are localized to the cell surface, have important roles in cell migration, and are active in the pericellular region. To identify peptides primarily cleaved by membrane-type proteases, an approach is developed that couples proteomic identification of candidate peptides with mass spectrometry-based functional assays to quantify degradation. The target sequence is then optimized using a split-and-pool synthesis to generate over 300 peptide variants to improve degradation behavior. The optimized peptide, KLVADLMASAE, shows reduced degradation by soluble proteases, while enabling endothelial and stem cell spreading and viability comparable to PanMMP hydrogels. KLVADLMASAE-crosslinked hydrogels have reduced crosslinker degradation, are stiffer during culture, and exhibit less macroscopic degradation after 14 days of culture than PanMMP gels. The performance of KLVADLMASAE-crosslinked gels is significantly improved from the initial peptide target, validating this functional high-throughput approach for identifying peptides that control matrix degradation.