连续固定化双酶反应器的制备及应用
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- 英文论文题名：Preparation and Applications of Continuous Bi-enzymatic reator
- 论文作者：张凌怡 ; 上官露露 ; 王兵兵 ; 王舒磊
- 英文论文作者：Lingyi Zhang ; L.L.Shangguan ; B.B.Wang ; S.L.Wang ; Shanghai Key Laboratory of Functional Materials Chemistry ; East China University of Science and Technology
- 年：2016
- 作者机构：华东理工大学上海市功能性材料化学重点实验室;
- 论文关键词：蛋白质组学 ; 固定化酶反应器 ; 连续双酶酶解 ; 胰蛋白酶 ; 糜蛋白酶
- 会议召开时间：2016-07-01
- 会议录名称：中国化学会第30届学术年会摘要集-第二十三分会：复杂样品分离分析
- 语种：中文
- 分类号：Q814.2
- 学会代码：ZGHY
- 会议名称：中国化学会第30届学术年会-第二十三分会 ; 复杂样品分离分析
- 会议地点：中国辽宁大连
- 主办单位：中国化学会
- 学会名称：中国化学会
- 页数：2
- 文件大小：177k
- 原文格式：D
- 会议级别：全国
摘要
发展高效快速的蛋白质酶解方法有助于推动蛋白质组学的深入研究。本文以包埋SBA-15纳米棒的有机无机杂化整体材料为载体,制备了连续固定化双酶酶反应器,并将其应用于复杂蛋白质样品酶解与分析。以包埋SBA-15-NH_2纳米材料的有机-无机杂化整体材料为载体,采用共价键合的固定化方法,将胰蛋白酶和糜蛋白酶分别固定在整体基质上,制备了两种单酶反应器,再将这两种单酶反应器按一定的比例偶联,形成连续双酶反应器。以碳酸酐酶和牛血清白蛋白作为标准样品,对酶反应器中两种酶含量以及酶解顺序对酶解效果的影响进行了考察,最终选择酶含量比为1:1,且先经胰蛋白酶酶解后由糜蛋白酶酶解的双酶酶解模式作为最佳的酶解方式。利用实际鼠肝蛋白提取样品,对两种串联双酶反应器(TCR/TCR)的酶解效率和酶解特征进行评价,其中串联双酶反应器(TCR)酶解得到1091种蛋白和5071条独立肽段,效果最佳。
Development of an efficient and fast digestion method will promote the further research of proteomics.In this paper,continous bi-enzyamtic reactors were prepared based on mixed matrix of hybrid organic-inorganic monolith with SBA-15 nanoparticles embedded,and further applied for the high-throughput proteome analysis.A series bi-enzyamtic reactor was prepared by connecting tryptic and chymotryptic reactors in according to a certain proportion.Single enzymatic reactor was obtained by immobilized trypsin/chymotrypsin onto a mixed matrix of hybrid organic-inorganic monolith with SBA-15-NH_2 nanoparticles embedded by covalent bonding with glutaraldehyde as bridge reagent.By using carbonic anhydrase and bovine serum albumin as standard samples,the effect of the content ratio of the two enzymes in the series bi-enzyamtic reactor and the order of digestion were investigated.Finally,digestions of rat liver proteins by seires bi-enzyamtic reactors(TCR/CTR) were performed and evaluated and 1091 proteins and 5071 peptides were identified.
Development of an efficient and fast digestion method will promote the further research of proteomics.In this paper,continous bi-enzyamtic reactors were prepared based on mixed matrix of hybrid organic-inorganic monolith with SBA-15 nanoparticles embedded,and further applied for the high-throughput proteome analysis.A series bi-enzyamtic reactor was prepared by connecting tryptic and chymotryptic reactors in according to a certain proportion.Single enzymatic reactor was obtained by immobilized trypsin/chymotrypsin onto a mixed matrix of hybrid organic-inorganic monolith with SBA-15-NH_2 nanoparticles embedded by covalent bonding with glutaraldehyde as bridge reagent.By using carbonic anhydrase and bovine serum albumin as standard samples,the effect of the content ratio of the two enzymes in the series bi-enzyamtic reactor and the order of digestion were investigated.Finally,digestions of rat liver proteins by seires bi-enzyamtic reactors(TCR/CTR) were performed and evaluated and 1091 proteins and 5071 peptides were identified.
引文
[1]Shangguan,L.L.,Zhang,L.Y.,Xiong,Z.C.,Ren,J.,et al.J.Chromatogr.A.2015,1388:158.
[2]Zhang,Z.D.,Zhang,L.Y.,Zhang,C.G.,Zhang,W.B.Talanta 2014,119:485-491.
[2]Zhang,Z.D.,Zhang,L.Y.,Zhang,C.G.,Zhang,W.B.Talanta 2014,119:485-491.