Liquid chromatographic analysis of phosphoamino acids at femtomole level using chemical derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate

Phosphorylation of amino acid residues in proteins plays a major role in biological systems. In this paper, a reversed-phase high performance liquid chromatographic (HPLC) method based on chemical derivatization has been described for the separation and quantification of phosphoamino acids at femtomole level, using fluorimetric detection (FLD). The protocol involved pre-column derivatization of phosphoamino acids with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) and subsequent separation on ZORBAX Eclipse XDB-C8 column. Several experimental factors that influenced derivatization and separation were carefully investigated. The derivatization was performed at 40 °C for 40 min in borate buffer (pH 8.5). Under the optimum conditions, phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) were satisfactorily separated in 8 min. The detection limits (signal-to-noise ratio = 3) for the phosphoamino acids could reach 10–20 fmol, which was the lowest value reported for HPLC methods and comparable to those obtained by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection methods. The proposed method has been validated and used to characterize the phosphoamino acids in the hydrolyzed phosphorylated protein samples. The results clearly demonstrated the potential of this technique to study phosphoamino acids as well as provided a new analytical methodology that should be applicable to the study of phosphorylation of protein in biological system.