The development of in situ methods for the analysis and visualization of enzyme activity is of paramount importance in drug discovery, research, and development. In this work, the functionalized and array patterned indium tin oxide (ITO) glass slides were fabricated by non-covalent immobilization of amphipathic phospholipid-tagged peptides encompassing the thrombin cleavage site on steric acid-modified ITO slides. The fabricated peptide arrays provide 60 spots per slide, and are compatible with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) measurement, free matrix peak interference, and tolerance to repeated aqueous washing. The peptide arrays were used for the investigation of thrombin activity and screening for its potential inhibitors. The thrombin activity and its Michaelis-Menten constant (K_m) for immobilized peptide substrate was determined using developed MALDI MS peptide array. To investigate the applicability and effectiveness of peptide arrays, the anti-thrombin activity of grape seed proanthocyanidins with different degrees of polymerization (DP) was monitored and visualized. MALDI MS imaging results showed that the fractions of proanthocyanidins with the mean DP of 4.61–6.82 had good thrombin inhibitory activity and their half-maximal inhibitory concentration (IC₅₀) were below 10 μg/mL. Therefore, the developed peptide array is a reliable platform for the discovery of natural thrombin inhibitors.

原地开发酶活性的分析和可视化方法在药物发现、研究和开发中至关重要。在这项工作中，功能化和阵列图案化的氧化铟锡 (ITO) 载玻片是通过非共价固定两亲性磷脂标记的肽（包括硬脂酸修饰的 ITO 载玻片上的凝血酶切割位点）制造的。制造的肽阵列每张载玻片提供 60 个点，并且与基质辅助激光解吸/电离质谱 (MALDI MS) 测量、无基质峰干扰以及对重复水洗的耐受性兼容。肽阵列用于研究凝血酶活性和筛选其潜在抑制剂。凝血酶活性及其 Michaelis-Menten 常数 (K _m) 使用开发的 MALDI MS 肽阵列测定固定肽底物。为了研究肽阵列的适用性和有效性，对不同聚合度 (DP) 的葡萄籽原花青素的抗凝血酶活性进行了监测和可视化。MALDI MS成像结果显示，平均DP为4.61-6.82的原花青素组分具有良好的凝血酶抑制活性，其半数最大抑制浓度（IC ₅₀）低于10 μg/mL。因此，开发的肽阵列是发现天然凝血酶抑制剂的可靠平台。