title: Top-down sequencing of O-glycoproteins by in-source decay matrix-assisted laser desorption ionization mass spectrometry for glycosylation site analysis.
abstract: The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17-18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154.
authors.affiliation: Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Köln, Germany. franz.hanisch@uni-koeln.de
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000737:chemistry / Q000378:metabolism; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D060748:Fetuins; D006020:Glycopeptides / Q000032:analysis; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D000509:alpha-Fetoproteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001212:Asialoglycoproteins; D002364:Caseins; D060748:Fetuins; D006020:Glycopeptides; D006023:Glycoproteins; D018396:Mucin-1; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin