Pugsley Haley R, Swearingen Kristian E, Dovichi Norman J
Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA.
J Chromatogr A. 2009 Apr 10;1216(15):3418-20. doi: 10.1016/j.chroma.2009.02.006. Epub 2009 Feb 10.
A number of algorithms have been developed to correct for migration time drift in capillary electrophoresis. Those algorithms require identification of common components in each run. However, not all components may be present or resolved in separations of complex samples, which can confound attempts for alignment. This paper reports the use of fluorescein thiocarbamyl derivatives of amino acids as internal standards for alignment of 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ)-labeled proteins in capillary sieving electrophoresis. The fluorescein thiocarbamyl derivative of aspartic acid migrates before FQ-labeled proteins and the fluorescein thiocarbamyl derivative of arginine migrates after the FQ-labeled proteins. These compounds were used as internal standards to correct for variations in migration time over a two-week period in the separation of a cellular homogenate. The experimental conditions were deliberately manipulated by varying electric field and sample preparation conditions. Three components of the homogenate were used to evaluate the alignment efficiency. Before alignment, the average relative standard deviation in migration time for these components was 13.3%. After alignment, the average relative standard deviation in migration time for these components was reduced to 0.5%.
已经开发出多种算法来校正毛细管电泳中的迁移时间漂移。这些算法需要识别每次运行中的共同组分。然而，在复杂样品的分离中，并非所有组分都可能存在或得到分离，这可能会使对齐的尝试变得混乱。本文报道了使用氨基酸的荧光素硫代氨基甲酰衍生物作为内标，用于在毛细管筛分电泳中对3-(2-呋喃甲酰基)喹啉-2-甲醛(FQ)标记的蛋白质进行对齐。天冬氨酸的荧光素硫代氨基甲酰衍生物在FQ标记的蛋白质之前迁移，精氨酸的荧光素硫代氨基甲酰衍生物在FQ标记的蛋白质之后迁移。这些化合物被用作内标，以校正细胞匀浆分离过程中两周内迁移时间的变化。通过改变电场和样品制备条件故意操纵实验条件。使用匀浆的三种组分来评估对齐效率。对齐前，这些组分迁移时间的平均相对标准偏差为13.3%。对齐后，这些组分迁移时间的平均相对标准偏差降至0.5%。
