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首页> 外文期刊>Journal of chromatography, A: Including electrophoresis and other separation methods >A one-step preparation method of monolithic enzyme reactor for highly efficient sample preparation coupled to mass spectrometry-based proteomics studies
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A one-step preparation method of monolithic enzyme reactor for highly efficient sample preparation coupled to mass spectrometry-based proteomics studies

机译：一种用于高效样品制备的整体式酶反应器的一步制备方法，结合基于质谱的蛋白质组学研究

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Mass spectrometry (MS) coupled to sample preparation and separation techniques has become a primary tool for proteomics studies. However, due to sample complexity, it is often challenging to achieve fast and efficient sample preparation prior to MS analysis. In recent decades, monolithic materials have been developed not only as chromatographic media, but also as efficient solid supports for immobilizing multiple types of affinity reagents. Herein, the N-acryloxysuccinimide-co-acrylamide-co-N,N'-methylenebisacrylamide (NAS-AAm-Bis) monolith was fabricated within silanized 200 mu m i.d. fused-silica capillaries and was used as an immobilized enzyme reactor (IMER). The column was conjugated with trypsin/Lys-C and Lys-N enzymes to allow enzymatic digestions to occur while protein mixture was loaded onto the IMER column followed by MS-based proteomics analysis. Similar MS signal and protein sequence coverage were observed using protein standard bovine serum albumin (BSA) compared to in-solution digestion. Furthermore, mouse serum, yeast, and human cell lysate samples were also subjected to enzymatic digestion by both IMER (in seconds to minutes) and conventional in solution digestion (overnight) for comparison in large-scale proteomics studies. Comparable protein identification results obtained by the two methods highlighted the potential of employing NAS-based IMER column for fast and highly efficient sample preparation for MS analysis in proteomics studies. (C) 2015 Elsevier B.V. All rights reserved.
机译：质谱（MS）与样品制备和分离技术的结合已成为蛋白质组学研究的主要工具。但是，由于样品的复杂性，在MS分析之前实现快速，高效的样品制备通常具有挑战性。在最近的几十年中，整体材料不仅被开发为色谱介质，而且还被开发为固定多种亲和试剂的有效固体载体。本文中，在硅烷化的200μmi.d.内制备了N-丙烯酰氧基琥珀酰亚胺-共丙烯酰胺-co-N，N′-亚甲基双丙烯酰胺（NAS-AAm-Bis）整料。熔融石英毛细管，并用作固定化酶反应器（IMER）。该柱与胰蛋白酶/ Lys-C和Lys-N酶偶联，可在将蛋白质混合物上样至IMER柱上时进行酶消化，然后进行基于MS的蛋白质组学分析。与溶液中消化相比，使用蛋白质标准牛血清白蛋白（BSA）观察到了相似的MS信号和蛋白质序列覆盖范围。此外，还通过IMER（数秒至数分钟）和常规溶液消化（过夜）对小鼠血清，酵母和人细胞裂解液样品进行了酶消化，以用于大规模蛋白质组学研究。通过两种方法获得的可比的蛋白质鉴定结果突显了使用基于NAS的IMER色谱柱为蛋白质组学研究中的MS分析快速高效制备样品的潜力。 （C）2015 Elsevier B.V.保留所有权利。

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