期刊
RAPID COMMUNICATIONS IN MASS SPECTROMETRY
卷 18, 期 15, 页码 1741-1748出版社
WILEY
DOI: 10.1002/rcm.1546
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We explored matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight (TOF/TOF) mass spectrometry for the analysis of N-glycosylated peptides, using horseradish peroxidase (HRP) as a test case. Two different types of cleavage were observed in the TOF/TOF fragmentation spectra: Firstly, cleavages of peptide bonds yielded fragments with the attached N-glycans staying intact, thus revealing information on peptide sequence and glycan attachment site. Secondly, fragmentation of the glycan moiety was characterized by cleavage of glycosidic bonds as well as a X-0,X-2-ring fragmentation of the innermost N-acetylglucosamine of the chitobiose core. Loss of the complete N-glycan moiety occurred by cleavage of both the N-glycosidic bond and the side-chain amide group of the N-glycosylated asparagine, yielding a characteristic peak doublet with a mass difference of 17 Da, which revealed the individual masses of the N-glycan and the peptide moiety. Analysis of a HRP tryptic digest at the sub-picomole level allowed the characterization of various N-glycosylated peptides including those with internal disulfide linkages, a glycopeptide linked via a disulfide bond to another peptide, and a 5 kDa glycopeptide carrying two N-glycans. The potential of our approach was illustrated by the detection of the following novel features of HRP glycosylation: W The conjugation of a xylosylated trimannosyl N-glycan without core-fucosylation to site Asn(316), showing for the first time unambiguously the occupation of this site; and (ii) A disaccharide N-acetylhexosamine(1)deoxyhexose, attached to N-glycosylation sites Asn(285) and Asn(298), which might represent a Fuc(alpha1-3)GlcNAc- moiety arising from the processing of N-glycans by a horse-radish endoglycosidase during biosynthesis of HRP. Copyright (C) 2004 John Wiley Sons, Ltd.
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