Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid-silica hybrid monolith as matrix for on-line protein digestion
Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid-silica hybrid monolith as matrix for on-line protein digestion
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DOI：
10.1016/j.chroma.2014.10.067
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发表时间：
2014-12-05
影响因子：
4.1
通讯作者：
Zhang, Yukui
中科院分区：
文献类型：
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作者：
Yuan, Huiming;Zhang, Lihua;Zhang, Yukui
本工作开发了一种新型的有机-硅胶杂化整体柱固定化酶反应器。以过硫酸铵为热引发剂，通过四甲氧基硅烷和乙烯基三甲氧基硅烷的缩聚反应，甲基丙烯酸和乙烯基基团在预缩聚硅氧烷上的原位共聚，采用一步一锅法制备了整体式载体。然后用N-(3-二甲氨基丙基)-N‘-乙基碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)活化，然后支化聚乙烯亚胺(PEI)以改善其亲水性。最后，经EDC和NHS活化后，将胰酶共价固定在整体载体上。通过对牛血清白蛋白(BSA)和肌红蛋白的顺序消化和MALDI-TOF-MS分析，对该微反应器的性能进行了评价。与传统的溶液内消化相比，不仅获得了更高的BSA(74+/-1.4%比59.5+/-2.7%，n=6)和肌红蛋白(93+/-3%比81+/-4.5%，n=6)的序列覆盖率，而且消化时间从24h缩短到2.5min，证明了这种Imer的高消化效率。对这两种蛋白质在Imer上的携带情况进行了研究，在肌红蛋白酶解的质谱图中没有发现来自BSA的多肽，这归因于我们开发的整体载体具有良好的亲水性。此外，蛋白质消化的动态浓度范围被证明为四个数量级，并且Imer至少可以连续使用7天。此外，将这种Imer与纳米RPLC-ESI/MS/MS偶联，对从大肠杆菌中提取的蛋白质进行了分析。与以往报道的基于硅胶杂化整体柱的Imer和传统的溶液中Imer相比，我们开发的Imer虽然鉴定的蛋白质数量相似，但鉴定的差异肽数分别提高了7%和25%，有利于提高蛋白质鉴定的可靠性。进一步将Imer与二维纳米高效液相色谱-MS/MS联用系统用于分析低转移率的肝细胞癌细胞蛋白质提取物，共鉴定出3000多个蛋白质组，仅从Imer残基中鉴定出46个蛋白质组。所有这些结果表明，这种基于Imer的混合型整体柱在高通量和高置信度蛋白质组分析中具有很大的应用前景。(C)2014爱思唯尔B.V.保留所有权利。
In this work, a novel kind of organic-silica hybrid monolith based immobilized enzymatic reactor (IMER) was developed. The monolithic support was prepared by a single step "one-pot" strategy via the polycondensation of tetramethoxysilane and vinyltrimethoxysilane and in situ copolymerization of methacrylic acid and vinyl group on the precondensed siloxanes with ammonium persulfate as the thermal initiator. Subsequently, the monolith was activated by N-(3-dimethylaminopropyl) - N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), followed by the modification of branched polyethylenimine (PEI) to improve the hydrophilicity. Finally, after activated by EDC and NHS, trypsin was covalently immobilized onto the monolithic support. The performance of such a microreactor was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin, followed by MALDI-TOF-MS analysis. Compared to those obtained by traditional in-solution digestion, not only higher sequence coverages for BSA (74 +/- 1.4% vs. 59.5 +/- 2.7%, n = 6) and myoglobin (93 +/- 3% vs. 81 +/- 4.5%, n = 6) were obtained, but also the digestion time was shortened from 24 h to 2.5 min, demonstrating the high digestion efficiency of such an IMER. The carry-over of these two proteins on the IMER was investigated, and peptides from BSA could not be found in mass spectrum of myoglobin digests, attributed to the good hydrophilicity of our developed monolithic support. Moreover, the dynamic concentration range for protein digestion was proved to be four orders of magnitude, and the IMER could endure at least 7-day consecutive usage. Furthermore, such an IMER was coupled with nano-RPLC-ESI/MS/MS for the analysis of extracted proteins from Escherichia coli. Compared to formerly reported silica hybrid monolith based IMER and the traditional in-solution counterpart, by our developed IMER, although the identified protein number was similar, the identified distinct peptide number was improved by 7% and 25% respectively, beneficial to improve the reliability of protein identification. The IMER was further online integrated with two-dimensional nano-HPLC-MS/MS system for the analysis of protein extracts from hepatocellular carcinoma (HCC) cells with low metastasis rate, and more than 3000 protein groups were identified, with only 46 proteins identified from the residues of the IMER. All these results demonstrated that such a hybrid monolith based IMER would be of great promise in the high throughput and high confidence proteome analysis. (C) 2014 Elsevier B.V. All rights reserved.