Abstract A novel immobilized enzyme reactor (IMER) based on trimethylolpropane trimethacrylate (TRIM) organic monolith was prepared by “thiol-ene” click reaction. The IMERs were made by fabricating TRIM monolith core in advance. There were residual double bonds on the surface of TRIM monolith, in which click reaction can be performed with the free thiol groups of trypsin at room temperature to bond the trypsin on the monolithic column without additionally introducing an active functional group. The enzyme activity was characterized by kinetic parameters Km and Vmax and determined by off-line chromatography. The performance of the IMERs was evaluated by digesting standard protein BSA and compared with in-solution digestion method. By using IMER 5 min, the sequence coverage rate of BSA was 79.41%, which was similar to that in-solution digestion for 12 h (82.7%). The applicability of the IMERs in complex samples was assessed by digesting crude protein extracts from egg white and mouse liver, identifying 28 (104 peptides) and 843 (3916 peptides) proteins, respectively. All results indicated that the IMERs have great potential in high-throughput proteomics analysis.

中文翻译：

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基于三羟甲基丙烷三甲基丙烯酸酯有机整体基质通过“硫醇-烯”点击反应的坚固的固定化酶反应器

摘要 通过“硫醇-烯”点击反应制备了一种基于三羟甲基丙烷三甲基丙烯酸酯（TRIM）有机整料的新型固定化酶反应器（IMER）。IMER 是通过预先制造 TRIM 整体核心制成的。TRIM整体柱表面有残留的双键，可以在室温下与胰蛋白酶的游离巯基进行点击反应，将胰蛋白酶键合在整体柱上，而无需额外引入活性官能团。酶活性由动力学参数 Km 和 Vmax 表征，并通过离线色谱法测定。通过消化标准蛋白质 BSA 评估 IMER 的性能，并与溶液中消化方法进行比较。使用IMER 5 min，BSA的序列覆盖率为79.41%，这类似于溶液中消化 12 小时 (82.7%)。通过消化来自蛋清和小鼠肝脏的粗蛋白提取物来评估 IMER 在复杂样品中的适用性，分别鉴定了 28（104 个肽段）和 843 个（3916 个肽段）蛋白质。所有结果表明，IMERs 在高通量蛋白质组学分析中具有巨大的潜力。