Fast and highly efficient digestion of proteins is essential for high-throughput proteomic analysis. Herein, a facile approach was developed for self-assembly preparation of trypsin-immobilized capillary monolithic column and its application as an immobilized enzyme microreactor (IMER) for fast and highly efficient proteolysis was described. The performance of the trypsin-immobilized monolithic enzyme microreactor was evaluated by in-situ digestion of model proteins. The results showed that the trypsin-immobilized monolithic enzyme microreactor had much higher tryptic digestion efficiency than the free trypsin in solution, where the coverage of peptide sequences by mass spectrometry (MS)-based analysis could bear comparison with the free one, while the digestion time was dramatically shortened from 12 h to 16 s. Furthermore, the trypsin-immobilized monolithic enzyme microreactor also exhibited good practicability to complex human serum sample, in which the total of 45 peptides from human serum albumin (HSA) matched with sequence coverage of 75% were precisely identified. The successful application demonstrated the promising potential of the trypsin-immobilized capillary monolithic column as the IMER in high-throughput proteomic analysis.

快速高效的蛋白质消化对于高通量蛋白质组学分析至关重要。在本文中，开发了一种简便的方法，用于胰蛋白酶固定的毛细管整体柱的自组装制备，并描述了其作为固定的酶微反应器（IMER）进行快速高效的蛋白水解的应用。通过原位评估胰蛋白酶固定的整体式酶微反应器的性能消化模型蛋白质。结果表明，固定化胰蛋白酶的整体式酶微反应器比溶液中的游离胰蛋白酶具有更高的胰蛋白酶消化效率，其中基于质谱（MS）的分析对肽序列的覆盖范围可以与游离胰蛋白酶进行比较，而消化时间从12 h大大缩短到16 s。此外，固定有胰蛋白酶的整体式酶微反应器对复杂的人血清样品也显示出良好的实用性，其中精确鉴定了来自人血清白蛋白（HSA）的45种肽，其序列覆盖率为75％。成功的应用证明了胰蛋白酶固定的毛细管整体柱作为IMER在高通量蛋白质组学分析中的潜在潜力。