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Electron transfer dissociation of N-glycopeptides: loss of the entire N-glycosylated asparagine side chain

Authors

  • M. Isabel Catalina,

    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands
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  • Carolien A. M. Koeleman,

    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands
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  • André M. Deelder,

    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands
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  • Manfred Wuhrer

    Corresponding author
    1. Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands
    • Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
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Abstract

The recently introduced electron transfer dissociation (ETD) technique opens new possibilities for the structural characterization of glycoproteins at the glycopeptide level. In this report, we investigate the ETD mass spectra of tryptic N-glycopeptides of the model glycoprotein horseradish peroxidase (HRP). Multiply protonated N-glycopeptides obtained by electrospray ionization were subjected to ETD. Fragment ions obtained by ETD were further analyzed by collision-induced dissociation (CID) (MS3) for their unambiguous structural assignment. The following fragmentation features were revealed: (1) c- and z-type peptide backbone cleavages were observed with retention of the intact glycan moiety revealing peptide sequence, glycan attachment site, and glycan mass; (2) to a lesser extent, glycosidic bond cleavages were registered with retention of the intact peptide sequence; and (3) a range of amino acid side chain losses did occur. Remarkably, the loss of the complete N-glycosylated asparagine side chain was observed. This loss of the glycan-modified side chain helps with the structural characterization of glycopeptides by allowing the facile deduction and verification of the glycan mass and the nature of the amino acid residue at the glycan attachment site. Importantly, informative ETD spectra were obtained in this study by reversed-phase nano-liquid chromatography (LC) coupled online to a radio-frequency (rf) quadrupole ion trap (QIT) mass spectrometer with alternating acquisition of CID and ETD mass spectra from an automatically selected set of precursors (data-dependent mode). Thus, our study brings nano-LC/QIT-MSn with CID and ETD to the fore as a powerful technique for glycoproteomics at the glycopeptide level. Copyright © 2007 John Wiley & Sons, Ltd.

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