An ultrasensitive chemical method for polysialic acid analysis.
An ultrasensitive method for analysis of polysialic acid (polySia) chains, using fluorescence-assisted high-performance liquid chromatography was developed. The new method is a substantial improvement of our earlier method in which the reducing terminal Sia residues of a homologous series of oligo/polySia hydrolytically released during derivatization reaction were simultaneously labeled with a fluorogenic reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB) in situ. We first studied extensively the stability of oligo/polySia in the acid (0.02 M trifluoracetic acid) used for 1,2-diamino-4,5-methylenedioxybenzene derivatization under various conditions of reaction time and temperature, analyzing the hydrolytic products by high-performance anion exchange chromatography with pulsed electrochemical detection (HPAEC-PED). Then we optimized the reaction conditions to minimize degradation of the parent polySia while maintaining high derivatization rate. Using a DNAPac PA-100 column rather than a MonoQ column, baseline resolution of polySia peaks up to DP 90 with a detection threshold of 1.4 femtomol per resolved peak was achieved. The new method was used to analyze the degree of polymerization of a polySia-containing glycopeptide fraction derived from embryonic chicken brain, and the results were compared with those obtained by HPAEC-PED.
Inoue
S
S
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, ROC.
Lin
S L
SL
Lee
Y C
YC
Inoue
Y
Y
D000818:Animals; D000837:Anion Exchange Resins; D001923:Brain Chemistry; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004563:Electrochemistry; D012680:Sensitivity and Specificity; D012794:Sialic Acids / Q000032:analysis*
D000837:Anion Exchange Resins; D012794:Sialic Acids; C021319:polysialic acid