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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.
Rapid Commun Mass Spectrom. 2004; 18(15):1741-8.RC

Abstract

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 Asn316, showing for the first time unambiguously the occupation of this site; and (ii) A disaccharide N-acetylhexosamine1deoxyhexose1 attached to N-glycosylation sites Asn285 and Asn298, 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.

Authors+Show Affiliations

Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. m.wuhrer@lumc.nlNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

15282773

Citation

Wuhrer, Manfred, et al. "Glycopeptide Analysis By Matrix-assisted Laser Desorption/ionization Tandem Time-of-flight Mass Spectrometry Reveals Novel Features of Horseradish Peroxidase Glycosylation." Rapid Communications in Mass Spectrometry : RCM, vol. 18, no. 15, 2004, pp. 1741-8.
Wuhrer M, Hokke CH, Deelder AM. Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation. Rapid Commun Mass Spectrom. 2004;18(15):1741-8.
Wuhrer, M., Hokke, C. H., & Deelder, A. M. (2004). Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation. Rapid Communications in Mass Spectrometry : RCM, 18(15), 1741-8.
Wuhrer M, Hokke CH, Deelder AM. Glycopeptide Analysis By Matrix-assisted Laser Desorption/ionization Tandem Time-of-flight Mass Spectrometry Reveals Novel Features of Horseradish Peroxidase Glycosylation. Rapid Commun Mass Spectrom. 2004;18(15):1741-8. PubMed PMID: 15282773.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Glycopeptide analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation. AU - Wuhrer,Manfred, AU - Hokke,Cornelis H, AU - Deelder,André M, PY - 2004/7/30/pubmed PY - 2004/12/16/medline PY - 2004/7/30/entrez SP - 1741 EP - 8 JF - Rapid communications in mass spectrometry : RCM JO - Rapid Commun Mass Spectrom VL - 18 IS - 15 N2 - 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 Asn316, showing for the first time unambiguously the occupation of this site; and (ii) A disaccharide N-acetylhexosamine1deoxyhexose1 attached to N-glycosylation sites Asn285 and Asn298, 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. SN - 0951-4198 UR - https://www.unboundmedicine.com/medline/citation/15282773/Glycopeptide_analysis_by_matrix_assisted_laser_desorption/ionization_tandem_time_of_flight_mass_spectrometry_reveals_novel_features_of_horseradish_peroxidase_glycosylation_ L2 - https://doi.org/10.1002/rcm.1546 DB - PRIME DP - Unbound Medicine ER -