Unraveling Performance: Proteomic Insights into Photo-Crosslinkable Biopolymer Properties

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 COL1α1 and COL1α2 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, for the first time, covalent crosslinks were identified and localized 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, enabling more precise tuning of material properties. This work contributes a foundational strategy for decoding bioactive hydrogel networks and supports improved design of GelMA scaffolds for applications such as osteogenesis.

甲基丙烯酸明胶（gelatin methacryloyl, GelMA）水凝胶尽管已被广泛应用于生物活性支架，但由于对其分子结构的调控能力不足，其合理设计仍受限于此。尤其在组织再生相关应用中，需在机械稳定性与细胞重塑微环境的能力之间达成精细平衡。本研究提出一种全新的整合工作流程，结合自下而上蛋白质组学分析、三维分子建模与结构表征，以阐明化学与物理修饰如何影响GelMA网络的形成。研究基于蛋白质组学数据构建三维模型，该数据鉴定了明胶来源的COL1α1与COL1α2链上的位点特异性甲基丙烯酰修饰。提升甲基丙烯酸酐当量（2.5倍与0.5倍当量）可提高取代度（GelMA 96与GelMA 34）与修饰频率，同时保留修饰的赖氨酸与精氨酸残基数量。圆二色谱（circular dichroism）与X射线衍射（X-ray diffraction）结果显示，高取代度的GelMA会以螺旋与转角结构为代价，提升β折叠结构的占比。经光交联后，研究构建了具有不同交联密度的网络，并首次通过蛋白质组学分析鉴定并定位了共价交联位点。该结构图谱揭示了多样的交联基序，并阐明了酶特异性如何影响检测结果。本研究所得见解将GelMA的化学性质与其交联行为及二级结构建立关联，可实现材料性能的更精准调控。本工作为解码生物活性水凝胶网络提供了基础性策略，可为骨生成等应用场景中的GelMA支架优化设计提供支撑。