GLYCOPEPTIDE PROFILING OF HUMAN URINARY ERYTHROPOIETIN BY MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY/

The site-specific 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 availabl e, a strategy combining optimal sensitivity and specificity was used, Erythropoietin was reduced, S-alkylated and digested with endoproteina se Lys C, The peptides were separated by reversed-phase high-performan ce liquid chromatography and the molecular masses of the peptides dete rmined by MALDI-MS, The peptides were identified by comparing the expe rimental masses with the masses predicted from the cDNA derived amino acid sequence, Glycopeptides were identified from the mass spectra bas ed or; the peak pattern caused By the glycan heterogeneity, They were further characterized after treatment with neuraminidase and endoprote ases, Ail N-glycosylation sites exhibited fucose-containing; complex-t ype glycans, The N-glycosylation sites at Asn(38) and Bsn(83) are main ly occupied by tetraantennary glycans, whereas Asn(24) is occupied by a mixture of bi-, tri- and tetraantennary glycans, A molecular mass gl ycoprofile for each glycosylation site was established based on the re lative peak intensities observed in the MALDI mass spectra of the desi alylated glycopeptides. (C) 1997 by John Wiley & Sons, Ltd.