Title :
Site-specific carbohydrate profiling of human
transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry
Abstract :
- Glycopeptides derived from a lysylendopeptidase digest of commercially available human transferrin were analyzed by nano-flow liquid chromatography/electrospray ionization mass spectrometry (LC/ ESI-MS), which permitted the carbohydrate profiles at Asn432 and Asn630 to be determined
- Both are located in a well-known motif for N-glycosylation, Asn-Xaa-Ser/Thr
- The contents of the carbohydrates at each site were significantly different from each other, and consisted of a variety of minor types of oligosaccharides in addition to the major one, a biantennary complex-type oligosaccharide
- Nano-flow ESI tandem mass spectrometry (MS/MS) of the glycopeptides (Cys421-Lys433 and Ile619-Lys646) containing these two sites yielded predominantly ions originating from the non-reducing termini (oxonium ions) and reducing terminus , resulting from cleavage of the glycosidic bonds of the carbohydrate moieties; this permitted the structural read-out of a small minority of the carbohydrate moieties
- In particular, the observation of oxonium ions at m/z 512.2 and 803.2 is useful for probing outer non-reducing terminal fucosylation , which represented carbohydrate structures consisting of Hex, dHex, and HexNAc, and NeuNAc, Hex, dHex, and HexNAc, respectively, from which the Lewis X structure (Galbeta1-4(Fucalpha1-3)GlcNAc) was readily deduced
- Moreover, fucosylation at the reducing-terminal GlcNAc fucosylation at the reducing-terminal GlcNAc (Fucalpha1-6GlcNAc) (Fucalpha1-6GlcNAc) specifically occurred at Asn630 , as demonstrated by treatment of the glycopeptides with alpha1-3/4-L-fucosidase