Carol Jeroen, Gorseling Maarten C J K, de Jong Camiel F, Lingeman Henk, Kientz Charles E, van Baar Ben L M, Irth Hubertus
Department of Analytical Chemistry and Applied Spectroscopy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Anal Biochem. 2005 Nov 1;346(1):150-7. doi: 10.1016/j.ab.2005.08.023. Epub 2005 Sep 7.
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%.
已开发出一种用于快速测定和鉴定蛋白质的多维分析方法。该方法基于对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，总分析时间缩短至不到100分钟。以牛血清白蛋白和肌红蛋白作为模型蛋白质，对消化时间和不同预处理步骤之间的接口条件等关键参数进行了优化。对该自动化系统进行了测试，以鉴定诸如霍乱毒素和葡萄球菌肠毒素B等传染病病原体。这通常导致通过约40%的总序列覆盖率进行阳性鉴定。
