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Recyclable trypsin immobilized magnetic nanoparticles based on hydrophilic polyethylenimine modification and their proteolytic characteristics†
Analytical Methods ( IF 2.7 ) Pub Date : 2017-12-22 00:00:00 , DOI: 10.1039/c7ay02418e Lingyi Zhang 1, 2, 3, 4, 5 , Bingbing Wang 1, 2, 3, 4, 5 , Shulei Wang 1, 2, 3, 4, 5 , Weibing Zhang 1, 2, 3, 4, 5
Analytical Methods ( IF 2.7 ) Pub Date : 2017-12-22 00:00:00 , DOI: 10.1039/c7ay02418e Lingyi Zhang 1, 2, 3, 4, 5 , Bingbing Wang 1, 2, 3, 4, 5 , Shulei Wang 1, 2, 3, 4, 5 , Weibing Zhang 1, 2, 3, 4, 5
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In this work, recyclable trypsin immobilized magnetic nanoparticles based on hydrophilic branched polyethylenimine (PEI) modification were synthesized under mild conditions. The amount of the immobilized enzyme was increased greatly by introducing branched PEI onto poly(methacrylic acid) (PMAA) modified Fe3O4 microspheres (denoted as Fe3O4@PMAA@PEI). Trypsin, a model enzyme for this study, was immobilized by Cu2+ with metal chelation to Fe3O4@PMAA@PEI. Compared with Fe3O4@PMAA nanoparticles, the abundant amino groups of the PEI layer not only increased the amount of the immobilized enzyme, but also improved the hydrophilicity of the material to avoid nonspecific protein adsorption. The performance of this material was evaluated by digesting bovine serum albumin (BSA), myoglobin and cytochrome C, followed by MALDI-TOF-MS analysis. Compared with in-solution digestion, not only the sequence coverage was improved (70.0 ± 3.1% vs. 40.0 ± 2.3% for BSA, 92.0 ± 2.4% vs. 81.0 ± 0.8% for myoglobin and 90.0 ± 3.0% vs. 56.0 ± 1.8% for cytochrome C), but also the digestion time was decreased from 24 h to 10 min. Furthermore, the relative standard deviation values (RSDs) of the sequence coverage of BSA were 3.0% for five consecutive digestions and 2.2% for particles from different batches (n = 3), showing good reproducibility. The Fe3O4@PMAA@PEI-trypsin can endure at least 7 times usage and still maintain good digestion performance after being stored at 4 °C for 25 days. When applied to complex samples, more proteins were identified from the extract of a rat liver than in-solution based digestion (582 vs. 496), with a shorter reaction time of 10 min.
中文翻译：
基于亲水性聚乙烯亚胺修饰的可回收胰蛋白酶固定的磁性纳米颗粒及其蛋白水解特性†
在这项工作中，在温和条件下合成了基于亲水性支链聚乙烯亚胺（PEI）修饰的可回收胰蛋白酶固定的磁性纳米颗粒。通过将支链PEI引入聚（甲基丙烯酸）（PMAA）修饰的Fe 3 O 4微球（表示为Fe 3 O 4 @ PMAA @ PEI），可大大增加固定化酶的量。胰蛋白酶，一种用于本研究的模型酶，通过与金属螯合的Cu 2+固定在Fe 3 O 4 @ PMAA @ PEI上。与Fe 3 O 4相比@PMAA纳米粒子，PEI层的丰富氨基基团不仅增加了固定化酶的量，而且还提高了材料的亲水性，从而避免了非特异性蛋白质的吸附。通过消化牛血清白蛋白（BSA），肌红蛋白和细胞色素C，然后进行MALDI-TOF-MS分析来评估该材料的性能。在溶液消化相比，不仅该序列覆盖率提高（70.0±3.1％对40.0±2.3％对BSA，92.0±2.4％对81.0±0.8％肌红蛋白和90.0±3.0％对比细胞色素C的含量为56.0±1.8％），但消化时间也从24小时减少到10分钟。此外，五次连续消化的BSA序列覆盖率的相对标准偏差值（RSD）为3.0％，来自不同批次（n = 3）的颗粒的2.2％相对标准偏差值，显示出良好的重现性。Fe 3 O 4 @ PMAA @ PEI-胰蛋白酶在4°C下储存25天后，可承受至少7倍的使用量并仍保持良好的消化性能。当应用于复杂样品时，与基于溶液的消化相比，从大鼠肝脏提取物中鉴定出的蛋白质更多（582 vs. 496），并且反应时间缩短了10分钟。
更新日期：2017-12-22
中文翻译：
基于亲水性聚乙烯亚胺修饰的可回收胰蛋白酶固定的磁性纳米颗粒及其蛋白水解特性†
在这项工作中，在温和条件下合成了基于亲水性支链聚乙烯亚胺（PEI）修饰的可回收胰蛋白酶固定的磁性纳米颗粒。通过将支链PEI引入聚（甲基丙烯酸）（PMAA）修饰的Fe 3 O 4微球（表示为Fe 3 O 4 @ PMAA @ PEI），可大大增加固定化酶的量。胰蛋白酶，一种用于本研究的模型酶，通过与金属螯合的Cu 2+固定在Fe 3 O 4 @ PMAA @ PEI上。与Fe 3 O 4相比@PMAA纳米粒子，PEI层的丰富氨基基团不仅增加了固定化酶的量，而且还提高了材料的亲水性，从而避免了非特异性蛋白质的吸附。通过消化牛血清白蛋白（BSA），肌红蛋白和细胞色素C，然后进行MALDI-TOF-MS分析来评估该材料的性能。在溶液消化相比，不仅该序列覆盖率提高（70.0±3.1％对40.0±2.3％对BSA，92.0±2.4％对81.0±0.8％肌红蛋白和90.0±3.0％对比细胞色素C的含量为56.0±1.8％），但消化时间也从24小时减少到10分钟。此外，五次连续消化的BSA序列覆盖率的相对标准偏差值（RSD）为3.0％，来自不同批次（n = 3）的颗粒的2.2％相对标准偏差值，显示出良好的重现性。Fe 3 O 4 @ PMAA @ PEI-胰蛋白酶在4°C下储存25天后，可承受至少7倍的使用量并仍保持良好的消化性能。当应用于复杂样品时，与基于溶液的消化相比，从大鼠肝脏提取物中鉴定出的蛋白质更多（582 vs. 496），并且反应时间缩短了10分钟。
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