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Rapid analysis of phosphoamino acids from phosvitin by near‐infrared cyanine 1‐(ε‐succinimydyl‐hexanoate)‐1′‐methyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine‐5,5′‐disulfonate potassium derivatization and polyacrylamide‐coated CE with LIF detection
Author(s) -
Fu NiNa,
Wang Hong,
Zhang HuaShan
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000540
Subject(s) - phosphoserine , chemistry , phosvitin , chromatography , derivatization , hydrolysis , polyacrylamide , high performance liquid chromatography , biochemistry , enzyme , protein kinase a , serine , polymer chemistry
A polyacrylamide‐coated CE method with LIF detection was developed for analyzing three phosphoamino acids including phosphotyrosine ( p ‐Tyr), phosphothreonine ( p ‐Thr), and phosphoserine ( p ‐Ser). A near‐infrared dye, 1‐(ε‐succinimydyl‐hexanoate)‐1′‐methyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine‐5,5′‐disulfonate potassium (MeCy5‐OSu) was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 6.1 min, using 10 mmol/L phosphate buffer (pH 4.0) containing 60 mmol/L SDS as running buffer. The highest derivatization efficiency was achieved in 0.2 mol/L borate buffer (pH 8.8) for 30 min at 30°C. Linearity of response was found in the range of 0.05–1 μmol/L. The correlation coefficients for these phosphoamino acids were from 0.9940 to 0.9976. The LODs for phosphotyrosine, phosphothreonine, and phosphoserine were about 6, 8, and 8 nmol/L, respectively. The proposed method has been successfully applied to the determination of phosphoamino acids in the hydrolysis sample of a phosphorylated phosvitin. Average recoveries for phosvitin sample were in the range of 94.0–98.0% and coefficients of variation ranged from 2.7 to 4.8%.