Structure elucidation of glycoproteins by direct nanoESI MS and MS/MS analysis of proteolytic glycopeptides
- 24 October 2007
- journal article
- Published by Wiley in Journal of Mass Spectrometry
- Vol. 42 (11), 1415-1421
- https://doi.org/10.1002/jms.1265
Abstract
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 (Man4 to Man9) 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 T280 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.Keywords
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