Efficient protein digestion using immobilized trypsin onto tannin modified Fe3O4 magnetic nanoparticles
Efficient protein digestion using immobilized trypsin onto tannin modified Fe3O4 magnetic nanoparticles
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DOI：
10.1016/j.colsurfb.2017.04.055
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发表时间：
2017-08-01
影响因子：
5.8
通讯作者：
Ozacar, Mahmut
中科院分区：
文献类型：
--
作者：
Atacan, Keziban;Cakiroglu, Bekir;Ozacar, Mahmut
采用溶剂热法制备了Fe3O4磁性纳米颗粒。以单宁(T)修饰的MNPs为载体，通过一种新的键合工艺，实现了一种商用胰酶的共价固定化。对所得纳米结构的形貌、结构、表面和磁性进行了全面的表征。制得的Fe3O4MNPs的室温饱和磁化值为60.18emu/g，单宁改性Fe3O4MNPs和单宁改性Fe3O4MNPs固定化胰酶的饱和磁化值分别为57.82emu/g和55.16emu/g，说明单宁包覆和胰酶固定化效果较好。通过对广泛使用的牛血清白蛋白(BSA)的酶消化，证实了固定化胰酶在蛋白质组学研究中的普遍适用性。将固定化胰酶分别在1min、5min和15min内对BSA进行胰酶消化。对15个S进行了微波辅助消化，用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)对消化后的蛋白质片段进行了鉴定，获得了令人满意的39个肽数，1min的序列覆盖率为84%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)分析表明，固定化胰酶能较好地消化牛血清白蛋白和蛋清蛋白。(C)2017爱思唯尔B.V.保留所有权利。
Fe3O4 magnetic nanoparticles (MNPs) were prepared via solvothermal method. A commercially available trypsin was covalently immobilized onto MNPs modified with tannin (T) via a novel binding process. The morphology, structure, surface and magnetic properties of the obtained nanostructures were characterized comprehensively. The Fe3O4 MNPs had a saturation magnetization value of 60.18 emu/g at room temperature, while the tannin modified Fe3O4 MNPs, and the trypsin immobilized on tannin modified-Fe3O4 MNPs possessed a saturation magnetization value of 57.82 emu/g and 55.16 emu/g, respectively, which indicated the decent tannin coating and trypsin immobilization. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of widely used bovine serum albumin (BSA). The immobilized trypsin was investigated by conducting the tryptic digestion of BSA within 1 min, 5 min and 15 min. Also, microwave-assisted digestion was carried out for 15 s. The digested protein fragments were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and a satisfactory peptide numbers of 39, and a superior sequence coverage of 84% for 1 min digestion were obtained. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed the satisfactory digestion of BSA and egg white proteins by immobilized trypsin. (C) 2017 Elsevier B.V. All rights reserved.