State Key Laboratory of Proteomics, National Center for Protein Science Beijing, Beijing Institute of Radiation Medicine, Beijing 102200, China; National Institute of Metrology, Beijing 100013, China.
Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, China.
Anal Chim Acta. 2017 Nov 22;994:19-28. doi: 10.1016/j.aca.2017.08.048. Epub 2017 Sep 20.
Mass spectrometry (MS) based bottom-up strategy is now the first choice for proteomics analysis. In this process, highly efficient and complete enzymatic degradation of protein samples is extremely important to achieve in-depth protein coverage and high-throughput protein profiling. However, conventional in-solution digestion suffer from long digestion time and enzyme autolysis that limit the protein sample processing throughput and identification accuracy. Here, we developed a novel type of magnetic metal organic frameworks (MOFs)-based immobilized enzyme reactor, FeO@DOTA-ZIF-90-trypsin. By introducing a stable chelator, 1,4,7,10-tetraazacyclododecane N,N',N″,N‴-tetraacetic acid (DOTA), onto the magnetic cores, the hybrid supporting matrix of the immobilized enzyme reactor FeO@DOTA-ZIF-90 has novel characteristics that include: i) favourable magnetic response (1.01 emu g) that makes the operation easy and convenient, ii) ultrahigh surface area (565.21 m g) and active sites that ensure high loading amounts and covalent linkage of enzyme, and iii) excellent structural and thermal stability that endows the immobilized enzyme reactor a prolonged lifespan. The performance of the magnetic MOFs-immobilized trypsin is first investigated using the standard protein, BSA, and the results showed that the immobilized enzyme reactor exhibits satisfactory digestion efficiency within only 1 min with the sequence coverage (80%) that is comparable or even better than that (70%) of the traditional 12 h-free trypsin digestion. To test the applicability of the magnetic MOFs-based immobilized enzyme reactor, protein samples extracted from 400 oocytes in mice were digested through the new immobilized enzyme reactor. In total, 8957 peptides corresponding to 1843 protein groups are identified, which are nearly of 40% and 67% increases in the number of identified proteins and peptides compared to using in-solution digestion with free proteases. Specifically, the identification of oocyte-specific proteins was critical in the discovery of and understanding the regulation mechanism of oocyte maturation. Thus, this synthetic procedure of FeO@DOTA-ZIF-90 provides a universal method for fabrication of magnetic MOFs materials, and the successful application of FeO@DOTA-ZIF-90-trypsin in efficient protein digestion for deeper proteome coverage will undoubtedly enlarge the uses for MOFs.
基于质谱（MS）的自上而下策略现在是蛋白质组学分析的首选。在这个过程中，高效且完全的酶解对实现深度蛋白质覆盖和高通量蛋白质分析非常重要。然而，传统的溶液内消化存在消化时间长和酶自解的问题，这限制了蛋白质样品处理的通量和鉴定的准确性。在这里，我们开发了一种新型的基于磁性金属有机骨架（MOFs）的固定化酶反应器，FeO@DOTA-ZIF-90-胰蛋白酶。通过在磁性核上引入稳定的螯合剂 1,4,7,10-四氮杂环十二烷 N,N',N″,N‴-四乙酸（DOTA），固定化酶反应器 FeO@DOTA-ZIF-90 的混合支撑基质具有以下新特性：i）有利的磁响应（1.01 emu g），使操作简单方便，ii）超高的表面积（565.21 m g）和活性位点，确保酶的高负载量和共价连接，iii）优异的结构和热稳定性，使固定化酶反应器具有更长的使用寿命。磁性 MOFs 固定化胰蛋白酶的性能首先使用标准蛋白 BSA 进行了研究，结果表明，固定化酶反应器在仅 1 分钟内即可达到令人满意的消化效率，其序列覆盖率（80%）与传统的 12 小时无胰蛋白酶消化相当，甚至更好。为了测试磁性 MOFs 固定化酶反应器的适用性，从 400 个小鼠卵母细胞中提取的蛋白质样品通过新的固定化酶反应器进行消化。总共鉴定到 8957 条肽段，对应 1843 个蛋白质组，与使用游离蛋白酶进行溶液内消化相比，鉴定到的蛋白质和肽段数量分别增加了近 40%和 67%。特别是，卵母细胞特异性蛋白质的鉴定对于发现和理解卵母细胞成熟的调控机制至关重要。因此，FeO@DOTA-ZIF-90 的这种合成方法为磁性 MOFs 材料的制备提供了一种通用方法，FeO@DOTA-ZIF-90-胰蛋白酶在高效蛋白质消化中的成功应用无疑将扩大 MOFs 的用途。
