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Liquid chromatographic analysis of phosphoamino acids at femtomole level using chemical derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate.
Anal Chim Acta. 2007 Oct 03; 601(1):118-24.AC

Abstract

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 degrees 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.

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Authors+Show Affiliations

Deng YH
Department of Chemistry, Wuhan University, Wuhan 430072, China.
Li RJ
No affiliation info available
Zhang HS
No affiliation info available
Du XL
No affiliation info available
Wang H
No affiliation info available

MeSH

Arabidopsis ProteinsChromatography, High Pressure LiquidPhosphoamino AcidsPhosphorylationProtein-Serine-Threonine KinasesProteinsReceptors, Cell SurfaceSensitivity and SpecificitySuccinimides

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17904477

Citation

Deng, Ying-Hua, et al. "Liquid Chromatographic Analysis of Phosphoamino Acids at Femtomole Level Using Chemical Derivatization With N-hydroxysuccinimidyl Fluorescein-O-acetate." Analytica Chimica Acta, vol. 601, no. 1, 2007, pp. 118-24.
Deng YH, Li RJ, Zhang HS, et al. Liquid chromatographic analysis of phosphoamino acids at femtomole level using chemical derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate. Anal Chim Acta. 2007;601(1):118-24.
Deng, Y. H., Li, R. J., Zhang, H. S., Du, X. L., & Wang, H. (2007). Liquid chromatographic analysis of phosphoamino acids at femtomole level using chemical derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate. Analytica Chimica Acta, 601(1), 118-24.
Deng YH, et al. Liquid Chromatographic Analysis of Phosphoamino Acids at Femtomole Level Using Chemical Derivatization With N-hydroxysuccinimidyl Fluorescein-O-acetate. Anal Chim Acta. 2007 Oct 3;601(1):118-24. PubMed PMID: 17904477.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Liquid chromatographic analysis of phosphoamino acids at femtomole level using chemical derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate. AU - Deng,Ying-Hua, AU - Li,Rong-Jun, AU - Zhang,Hua-Shan, AU - Du,Xiao-Lan, AU - Wang,Hong, Y1 - 2007/08/22/ PY - 2007/06/08/received PY - 2007/08/15/revised PY - 2007/08/15/accepted PY - 2007/10/2/pubmed PY - 2007/11/9/medline PY - 2007/10/2/entrez SP - 118 EP - 24 JF - Analytica chimica acta JO - Anal Chim Acta VL - 601 IS - 1 N2 - 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 degrees 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. SN - 1873-4324 UR - https://www.unboundmedicine.com/medline/citation/17904477/Liquid_chromatographic_analysis_of_phosphoamino_acids_at_femtomole_level_using_chemical_derivatization_with_N_hydroxysuccinimidyl_fluorescein_O_acetate_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2670(07)01386-4 DB - PRIME DP - Unbound Medicine ER -
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