Confirmation of peak assignments in capillary electrophoresis using immunoprecipitation. Application to D-aspartate measurements in neurons.
Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection is a powerful tool for analysis of samples ranging from tissue extracts to single cells. However, accurate peak identification in electropherograms is challenging when complex biological samples are analyzed, as often matching a migration time between an analyte and corresponding standard may be insufficient to confirm the peak's identity. A method which combines single-step immunoprecipitation and CE-LIF analysis for investigation of the chiral amino acids in single cells and small tissue samples is demonstrated. D-Aspartate (D-Asp) has been reported in the central nervous system of the invertebrate neurobiological model Aplysia californica. In order to confirm the identity of D-Asp signal in the complex electropherograms of nerve tissue extracts and individual neurons, anti-D-Asp serum, preincubated with L-Asp conjugate, is added to the sample. This selectively binds the free D-Asp, creating an antibody-antigen complex with a migration time similar to that of antibody alone, but not that of D-Asp. The complete disappearance of the putative D-Asp peak confirms its identity and validates that there are no other detectable analytes co-migrating with D-Asp in the electropherogram.
Miao
Hai
H
Department of Chemistry, University of Illinois, Champaign, School of Chemical Sciences, 600 South Mathews Avenue 63-5, Urbana, IL 61801, USA.
Rubakhin
Stanislav S
SS
Sweedler
Jonathan V
JV
D000818:Animals; D001048:Aplysia; D026603:D-Aspartic Acid / Q000032:analysis*; D019075:Electrophoresis, Capillary / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D009474:Neurons / Q000737:chemistry*
D026603:D-Aspartic Acid