Fluorescein thiocarbamyl amino acids as internal standards for migration time correction in capillary sieving electrophoresis.
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%.
Pugsley
Haley R
HR
Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA.
Swearingen
Kristian E
KE
Dovichi
Norman J
NJ
D000465:Algorithms; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D019793:Fluorescein / Q000737:chemistry*; D005663:Furans / Q000032:analysis; D006801:Humans; D011804:Quinolines / Q000032:analysis; D012015:Reference Standards
D005663:Furans; D011804:Quinolines; C063756:3-(2-furoyl)quinoline-2-carbaldehyde; D001224:Aspartic Acid; D001120:Arginine; D019793:Fluorescein