Site-specific carbohydrate profiling of human transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry.
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 (Fucalpha1-6GlcNAc) specifically occurred at Asn630, as demonstrated by treatment of the glycopeptides with alpha1-3/4-L-fucosidase.
Satomi
Yoshinori
Y
Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
Shimonishi
Yasutsugu
Y
Hase
Toshiharu
T
Takao
Toshifumi
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014168:Transferrin / Q000032:analysis* / Q000737:chemistry*
D006020:Glycopeptides; D014168:Transferrin