Kecskemeti Adam, Nagy Cynthia, Csosz Eva, Kallo Gergo, Gaspar Attila
Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen, Hungary.
Department of Biochemistry and Molecular Biology, University of Debrecen, Debrecen, Hungary.
Proteomics Clin Appl. 2017 Dec;11(11-12). doi: 10.1002/prca.201700055. Epub 2017 Jul 24.
The application of a newly developed microfluidic immobilized enzymatic reactor (IMER) designed to accelerate protein digestion in clinical samples is presented.
The IMER contains trypsin adsorbed on the porous surface of a PDMS microfluidic chip. Human tear with its relatively low volume and high protein content is collected and used for testing the digestion efficiency of the IMER. With the use of CZE peptide mapping, the efficiency and reproducibility of the reactor are investigated.
No significant difference is observed in the CZE peptide profiles of the same tear sample digested in-solution or via microfluidic IMER. LC-MS measurements show that the microfluidic IMER digestion enables the identification of more proteins compared to standard in-solution digestion and those proteins that are identified with both digestion methods have higher sequence coverage when digested with the IMER.
The proposed reactor is well-suited for rapid and efficient protein digestion and even eight digestions can be carried out simultaneously. The PDMS chip is inexpensive and easy to fabricate, thus its application can be an attractive alternative for proteomic related research.
介绍一种新开发的微流控固定化酶反应器（IMER）在临床样本中加速蛋白质消化的应用。
IMER包含吸附在PDMS微流控芯片多孔表面的胰蛋白酶。收集体积相对较小且蛋白质含量高的人眼泪用于测试IMER的消化效率。使用毛细管区带电泳肽图谱分析，研究反应器的效率和重现性。
在溶液中消化或通过微流控IMER消化的同一眼泪样本的毛细管区带电泳肽图谱中未观察到显著差异。液相色谱-质谱测量表明，与标准溶液消化相比，微流控IMER消化能够鉴定出更多蛋白质，并且两种消化方法都能鉴定出的蛋白质在用IMER消化时具有更高的序列覆盖率。
所提出的反应器非常适合快速高效的蛋白质消化，甚至可以同时进行八次消化。PDMS芯片价格低廉且易于制造，因此其应用对于蛋白质组学相关研究可能是一种有吸引力的替代方案。
