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Glycopeptide analysis by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation
Author(s) -
Wuhrer Manfred,
Hokke Cornelis H.,
Deelder André M.
Publication year - 2004
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1546
Subject(s) - chemistry , glycosidic bond , glycan , tandem mass spectrometry , glycosylation , peptide , matrix assisted laser desorption/ionization , fucosylation , glycopeptide , mass spectrometry , horseradish peroxidase , peptide bond , stereochemistry , chromatography , biochemistry , organic chemistry , glycoprotein , adsorption , desorption , antibiotics , enzyme
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 0,2 X‐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: (i) 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 1 attached to N‐glycosylation sites Asn 285 and Asn 298 , which might represent a Fuc( α 1‐3)GlcNAc‐ moiety arising from the processing of N‐glycans by a horse‐radish endoglycosidase during biosynthesis of HRP. Copyright © 2004 John Wiley & Sons, Ltd.