基于温敏型材料的固定化酶的制备及其在蛋白质组快速分析中的应用

蛋白质组学通过规模化鉴定、分析从细胞、组织或有机体中提取的蛋白质，从而获得蛋白表达、修饰、组成和定量的变化信息。在目前最为有效的“鸟枪法”蛋白质组学策略中，固定化酶试剂基质常用固相载体材料，该固定化酶试剂在酶解蛋白质时为异相体系，存在固液界面传质阻力和空间位阻，限制了酶解效率和样品处理通量。针对这一技术瓶颈，本研究利用温敏聚合物对外界温度变化的响应能力，制备了一种新型的基于可溶性温敏聚合物的固定化胰蛋白酶试剂。该固定化酶特有的温度敏感特性，使其具有“高温均相酶解，低温异相分离”的特色，且兼具酶切时间显著缩短、酶可重复利用的优势。 BSA 1 min固定化酶解产物肽段的氨基酸序列覆盖率可达94%，高于传统溶液酶解12 h所得覆盖率为(74%)。进一步将该固定化酶试剂应用于HeLa细胞全蛋白质组的酶解，其酶解效果与相同条件下溶液酶解12 h相当。该固定化酶试剂对复杂蛋白质的快速、高效酶解充分证明其在蛋白质组学研究中的应用潜力。

Preparation and Application of Novel Thermo-sensitive Matrix-based Immobilized Enzyme for Fast and Highly Efficient Proteome Study

By massively analyzing proteins extracted from cells, tissues and organisms using mass spectrometry, proteomics is capable of providing information about change in proteins expression, modification, composition and quantification. However, most immobilized enzymes used in mass spectrometry based "shotgun" proteomic strategy are prepared using solid materials as the immobilization matrix and digest the substrate proteins in heterogeneous system. The inherent mass transfer resistance in the solid-liquid interface and steric hindrance of the solid matrix limits the digestion efficiency and sample processing throughput. Here, we prepared a novel immobilized enzyme using soluble thermo-sensitive polymer as the matrix material by exploiting the thermo-responsive ability of the polymer to environmental temperature changes. The thermo-sensitive immobilized trypsin had the feature of “homogeneous digestion at high temperature and heterogeneous separation at low temperature” and the advantage of significantly shortened digestion time and recover and reuse of the enzyme. An amino acid sequence covering up to 94% in 1-min digestion was obtained, which was higher than that of 74% obtained by in-solution digestion for 12 h. Finally, the immobilized trypsin was successfully applied to fast and highly efficient digestion of complex proteome extracted from HeLa cell. The efficiency of immobilized digestion in 1 min was similar to that of solution digestion in 12 h, which sufficiently demonstrated the application potential of this thermo-sensitive immobilized trypsin in proteomics research.