Fluorescent-assisted detection of oligosialyl units in glycoconjugates

A highly sensitive chemical method to detect various types of oligo/polysialic acid (oligo/polySia) units in glycoconjugates, i.e., alpha2 --> 8-linked homo-oligo/polySia [N-acetylneuraminic acid (Neu5Ac), N-glcolylneuraminic acid (Neu5Gc), or 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid], alpha2 --> 8-linked heterodimers of Neu5Ac and Neu5Gc, alpha2 --> 9-linked homooligo/polyNeu5Ac, and alpha2 --> 5-Oglycolyl-linked homooligo/polyNeu5Gc, was developed with an alpha-keto acid-reactive fluorescent labeling reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB). Fluorescent labeled di- or oligoSia was separated and quantitated by fluorometric anion-exchange high-performance liquid chromatography (HPLC). As little as 13 fmol of Neu5Acalpha2 --> 8Neu5Ac was detectable by this method. When alpha2 --> 8-linked oligo/polyNeu5Ac with on average eight Neu5Ac residues was labeled with DMB, DMB derivatives of oligomers with only lower degrees of polymerization of 2 to 7, were detected, due to concomitant partial depolymerization of oligo/polySia chain with the derivatization. For glycoproteins and glycolipids, mild acid hydrolysis was performed to release oligoSia prior to DMB derivatization. The mild acid hydrolysis/fluorometric HPLC method was also applicable to glycoprotein samples blotted on the membrane.