Capillary electrophoresis of phosphorylated amino acids with fluorescence detection.
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 3.5mM NDA, 40 mM NaCN and 20mM 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 2.0mM beta-cyclodextrin (beta-CD) as the running buffer. The detection limits for phosphothreonine, phosphotyrosine and phosphoserine were 7.0 x 10(-9)M, 5.6 x 10(-9)M and 7.2 x 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 beta-casein as the analysis protein, this method was successfully validated.
Zhang
Li-Yao
LY
Key Laboratory of MOE for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
Sun
Meng-Xiang
MX
D002138:Calibration; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000737:chemistry; D009281:Naphthalenes / Q000737:chemistry; D019606:Phosphoamino Acids / Q000032:analysis*; D010766:Phosphorylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D047392:beta-Cyclodextrins / Q000737:chemistry
D002364:Caseins; D007202:Indicators and Reagents; D009281:Naphthalenes; D019606:Phosphoamino Acids; D047392:beta-Cyclodextrins; C027717:2,3-naphthalenedicarboxaldehyde; C031215:betadex