Structure elucidation of glycoproteins by direct nanoESI MS and MS/MS analysis of proteolytic glycopeptides.
Bovine ribonuclease B (RNAse B) and asialofetuin (FETUA) were subjected to in-capillary tryptic digest (Pohlentz et al. Proteomics. 2005, 5, 1758-1763) and the obtained glycopeptides were analyzed, respectively, by nanoelectrospray ionization mass spectrometry and collision-induced dissociation (CID) during the ongoing digest. For RNAse, B glycans of the high-mannose type (Man(4) to Man(9)) attached to either a tetra- or a hexapeptide containing the sole N-glycosylation site of the protein were detected. Glycopeptides derived from all three N-glycosylation sites of FETUA were observed, and the corresponding CID spectra proved the respective glycans to be oligosaccharides of the triantennary complex type. Moreover, an O-glycopeptide carrying Gal-GalNAc at T(280) could be unambiguously identified. An in-solution tryptic/chymotryptic digest of human transferrin (TRFE) was analyzed directly for glycopeptides subsequent to the addition of methanol and formic acid. Disialylated diantennary glycans were observed in glycopeptides of both N-glycosylation sites of TRFE. These results demonstrate the feasibility of direct structure determination of glycopeptides in proteolytic mixtures without any further refurbishment.
Henning
Stefanie
S
Institute for Medical Physics and Biophysics, University of Münster, Münster, Germany.
Peter-Katalinić
Jasna
J
Pohlentz
Gottfried
G
D000818:Animals; D001212:Asialoglycoproteins / Q000737:chemistry; D002240:Carbohydrate Sequence; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry; D060748:Fetuins; D006020:Glycopeptides / Q000032:analysis*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000032:analysis; D025942:Oligosaccharides, Branched-Chain / Q000032:analysis; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014168:Transferrin / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000737:chemistry
D001212:Asialoglycoproteins; D060748:Fetuins; D006020:Glycopeptides; D006023:Glycoproteins; D009844:Oligosaccharides; D025942:Oligosaccharides, Branched-Chain; D014168:Transferrin; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012259:Ribonuclease, Pancreatic; C504335:pancreatic ribonuclease B, bovine; D002918:Chymotrypsin; D014357:Trypsin