Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electro spray mass spectrometry
Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electro spray mass spectrometry
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DOI：
10.1016/j.ab.2005.08.023
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发表时间：
2005-11-01
影响因子：
2.9
通讯作者：
Irth, H
中科院分区：
文献类型：
--
作者：
Carol, J;Gorseling, MCJK;Irth, H
已经开发出一种用于快速测定和鉴定蛋白质的多维分析方法。该方法基于含蛋白质样品的尺寸排阻分级分离、随后的在线胰蛋白酶消化和脱盐以及反相高效液相色谱-电喷雾质谱检测。本系统将消化时间缩短至 20 分钟，并将总分析时间缩短至不到 100 分钟。使用牛血清白蛋白和肌红蛋白作为模型蛋白，对消化时间和不同预处理步骤之间的界面条件等关键参数进行了优化。该自动化系统经过测试，可鉴定霍乱毒素和葡萄球菌肠毒素 B 等传染病病原体。这通常会导致总序列覆盖率约为 40% 的阳性鉴定。 (C) 2005 Elsevier Inc. 保留所有权利。
A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography-electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%. (C) 2005 Elsevier Inc. All rights reserved.