Z. L. Wu, X. Huang, A. J. Burton, and K. A.D. Swift
ABSTRACT
We describe a novel technique for probing terminal glycans on glycoproteins. Our approach consists of in vitro incorporation of specially developed azido-monosaccharides and various recombinant glycosyltransferases and glycosidases, followed by detection via click chemistry and traditional chemiluminescence. The technique uses SDS-PAGE and protein transfer in a manner similar to Western blot. It is very specific and does not require expensive equipment such as mass spectrometry. To demonstrate its utility, we probed for both N- and O-linked sialoglycans. Using fetal bovine fetuin as an example, we demonstrated: (I) The non-reducing end Gal residues on both N-glycans and O-glycans on the protein are fully sialylated, while the O-linked GalNAc residues are not; (II) The protein contains abundant sialyl core-1 glycan; (III) The protein also contains sialyl Tn antigen. We also present a method for specifically probing high mannose glycans.
