Capillary electrophoresis of phosphorylated amino acids with fluorescence detection

Abstract

A rapid and sensitive capillary electrophoresis (CE) method coupled with fluorescence detection was developed for identification of protein phosphorylation by determination of phosphoamino acids. Naphthalene-2,3-dicarboxaldehyde (NDA), a fluorescence derivatization reagent, was used to label protein hydrolysate. The optimal derivatization reaction was performed with 3.5 mM NDA, 40 mM NaCN and 20 mM borate buffer (pH 10.0) for 15 min. The baseline separation of three phosphorylated amino acids could be obtained in less than 180 s with good repeatability by using 30 mM borate (pH 9.2) containing 2.0 mM β-cyclodextrin (β-CD) as the running buffer. The detection limits for phosphothreonine, phosphotyrosine and phosphoserine were 7.0 × 10 −9 M, 5.6 × 10 −9 M and 7.2 × 10 −9 M, respectively ( S/ N = 3). Also, the interference from other protein amino acids with large molar excess over that of phosphoamino acids was studied. With β-casein as the analysis protein, this method was successfully validated.