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Facile synthesis of magnetic metal organic framework for highly efficient proteolytic digestion used in mass spectrometry-based proteomics
Analytica Chimica Acta ( IF 6.2 ) Pub Date : 2017-11-01 , DOI: 10.1016/j.aca.2017.08.048
Rui Zhai , Yifeng Yuan , Fenglong Jiao , Feiran Hao , Xiang Fang , Yangjun Zhang , Xiaohong Qian

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, Fe3O4@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 Fe3O4@DOTA-ZIF-90 has novel characteristics that include: i) favourable magnetic response (1.01 emu g-1) that makes the operation easy and convenient, ii) ultrahigh surface area (565.21 m2 g-11) 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 Fe3O4@DOTA-ZIF-90 provides a universal method for fabrication of magnetic MOFs materials, and the successful application of Fe3O4@DOTA-ZIF-90-trypsin in efficient protein digestion for deeper proteome coverage will undoubtedly enlarge the uses for MOFs.

中文翻译:

用于基于质谱的蛋白质组学高效蛋白水解消化的磁性金属有机骨架的简便合成

基于质谱 (MS) 的自下而上策略现在是蛋白质组学分析的首选。在此过程中,蛋白质样品的高效完整酶促降解对于实现深入的蛋白质覆盖和高通量蛋白质分析极为重要。然而,传统的溶液内消化受到消化时间长和酶自溶的影响,这限制了蛋白质样品的处理通量和识别准确性。在这里,我们开发了一种新型的基于磁性金属有机框架 (MOF) 的固定化酶反应器 Fe3O4@DOTA-ZIF-90-胰蛋白酶。通过在磁芯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 Fe3O4@DOTA- ZIF-90 具有新颖的特性,包括:i) 良好的磁响应 (1.01 emu g-1),使操作简单方便,ii) 超高表面积 (565.21 m2 g-11) 和活性位点,确保高负载量和酶的共价连接,以及 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。首先使用标准蛋白质 BSA 研究了磁性 MOFs 固定化胰蛋白酶的性能,结果表明固定化酶反应器在 1 分钟内表现出令人满意的消化效率,序列覆盖率(80%)相当甚至更好比传统的 12 小时无胰蛋白酶消化 (70%) 高。为了测试基于磁性 MOFs 的固定化酶反应器的适用性,从小鼠 400 个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。总共鉴定了 8957 种肽,对应于 1843 个蛋白质组,与使用游离蛋白酶进行溶液内消化相比,鉴定的蛋白质和肽的数量增加了近 40% 和 67%。具体而言,卵母细胞特异性蛋白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,Fe3O4@DOTA-ZIF-90 的这种合成过程为磁性 MOFs 材料的制造提供了一种通用方法,而 Fe3O4@DOTA-ZIF-90-胰蛋白酶在高效蛋白质消化中的成功应用,以实现更深的蛋白质组覆盖无疑将扩大用于 MOF。鉴定出 8957 个肽段对应于 1843 个蛋白质组,与使用游离蛋白酶进行溶液内消化相比,鉴定出的蛋白质和肽段数量增加了近 40% 和 67%。具体而言,卵母细胞特异性蛋白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,Fe3O4@DOTA-ZIF-90 的这种合成过程为磁性 MOFs 材料的制造提供了一种通用方法,而 Fe3O4@DOTA-ZIF-90-胰蛋白酶在高效蛋白质消化中的成功应用,以实现更深的蛋白质组覆盖无疑将扩大用于 MOF。鉴定出 8957 个肽段对应于 1843 个蛋白质组,与使用游离蛋白酶进行溶液内消化相比,鉴定出的蛋白质和肽段数量增加了近 40% 和 67%。具体而言,卵母细胞特异性蛋白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,Fe3O4@DOTA-ZIF-90 的这种合成过程为磁性 MOFs 材料的制造提供了一种通用方法,而 Fe3O4@DOTA-ZIF-90-胰蛋白酶在高效蛋白质消化中的成功应用,以实现更深的蛋白质组覆盖无疑将扩大用于 MOF。具体而言,卵母细胞特异性蛋白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,Fe3O4@DOTA-ZIF-90 的这种合成过程为磁性 MOFs 材料的制造提供了一种通用方法,而 Fe3O4@DOTA-ZIF-90-胰蛋白酶在高效蛋白质消化中的成功应用,以实现更深的蛋白质组覆盖无疑将扩大用于 MOF。具体而言,卵母细胞特异性蛋白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,Fe3O4@DOTA-ZIF-90 的这种合成过程为磁性 MOFs 材料的制造提供了一种通用方法,而 Fe3O4@DOTA-ZIF-90-胰蛋白酶在高效蛋白质消化中的成功应用,以实现更深的蛋白质组覆盖无疑将扩大用于 MOF。
更新日期:2017-11-01
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