Structure-function relationship of methacryloyl-modified gelatin revealed by proteomics and 3D modeling

Rational design of gelatin methacryloyl (GelMA) hydrogels remains limited by insufficient control over their molecular architecture, despite their broad use in bioactive scaffolds. Especially for applications involving tissue regeneration, a delicate balance is needed between mechanical stability and the ability of cells to remodel their microenvironment. In this study, we present a novel integrative workflow combining bottom-up proteomic analysis, 3D molecular modeling, and structural characterization to elucidate how chemical and physical modifications influence GelMA network formation. A 3D model was built from proteomics data identifying site-specific methacryloyl modifications on gelatin-derived COL1A1 and COL1A2 chains. Increasing methacrylic anhydride equivalents (2.5 vs. 0.5 eq) raised the degree of substitution (GelMA 96 vs. GelMA 34) and the frequency of modification, while preserving the number of modified lysines and arginines. Circular dichroism and X-ray diffraction revealed that highly substituted GelMA showed increased β-strand formation at the expense of helices and turns. Upon photo-crosslinking, networks with different crosslink densities were created and, covalent crosslinks were identified and localized for the first time using proteomic analysis. This structural mapping demonstrated diverse crosslinking motifs and highlighted how enzyme specificity influences detection. The resulting insights connect GelMA chemistry to its crosslinking behavior and secondary structure, thereby enabling more precise tuning of material properties within this type-B GelMA system by linking synthesis parameters to experimentally measured molecular descriptors, rather than eliminating the intrinsic heterogeneity of gelatin. This work contributes a foundational strategy for decoding bioactive hydrogel networks and supports improved design of GelMA scaffolds for applications such as osteogenesis.