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Structure elucidation of glycoproteins by direct nanoESI MS and MS/MS analysis of proteolytic glycopeptides
Author(s) -
Henning Stefanie,
PeterKatalinić Jasna,
Pohlentz Gottfried
Publication year - 2007
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.1265
Subject(s) - chemistry , glycopeptide , glycoprotein , chromatography , biochemistry , antibiotics
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. Copyright © 2007 John Wiley & Sons, Ltd.