Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry

✍ Scribed by Rahbek-Nielsen, Henrik; Roepstorff, Peter; Reischl, Heinz; Wozny, Manfred; Koll, Hans; Haselbeck, Anton

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 597 KB
• Volume: 32
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/(sici)1096-9888(199709)32:9<948::aid-jms551>3.0.co;2-c

✦ Synopsis

The site-speciÐc glycan heterogeneity of human urinary erythropoietin was investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Owing to the small amount of protein available, a strategy combining optimal sensitivity and speciÐcity was used. Erythropoietin was reduced, S-alkylated and digested with endoproteinase Lys C. The peptides were separated by reversed-phase high-performance liquid chromatography and the molecular masses of the peptides determined by MALDI-MS. The peptides were identiÐed by comparing the experimental masses with the masses predicted from the cDNA derived amino acid sequence. Glycopeptides were identiÐed from the mass spectra based on the peak pattern caused by the glycan heterogeneity. They were further characterized after treatment with neuraminidase and endoproteases. All N-glycosylation sites exhibited fucose-containing complex-type glycans. The N-glycosylation sites at and are mainly Asn 38 Asn 83 occupied by tetraantennary glycans, whereas is occupied by a mixture of bi-, tri-and tetraantennary glycans. Asn 24 A molecular mass glycoproÐle for each glycosylation site was established based on the relative peak intensities observed in the MALDI mass spectra of the desialylated glycopeptides.

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