Volume 18, Issue 24 1800282
Research Article

The Fine Art of Destruction: A Guide to In-Depth Glycoproteomic Analyses—Exploiting the Diagnostic Potential of Fragment Ions

Marcus Hoffmann

Marcus Hoffmann

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany

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Markus Pioch

Markus Pioch

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany

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Alexander Pralow

Alexander Pralow

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany

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René Hennig

René Hennig

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany

glyXera GmbH, 39120 Magdeburg, Germany

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Robert Kottler

Robert Kottler

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany

glyXera GmbH, 39120 Magdeburg, Germany

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Udo Reichl

Udo Reichl

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany

Chair of Bioprocess Engineering, Otto von Guericke University Magdeburg, 39106 Magdeburg, Germany

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Erdmann Rapp

Corresponding Author

Erdmann Rapp

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, 39106 Magdeburg, Germany

glyXera GmbH, 39120 Magdeburg, Germany

Correspondence: Dr. Erdmann Rapp ([email protected])Search for more papers by this author
First published: 14 November 2018
Citations: 29

Abstract

The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro- and macroheterogeneity of glycoproteins. Here, combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of N- and O-glycopeptides is proposed. Using a set of four representative and biopharmaceutically relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonuclease B), the benefits and limitations of the developed workflow are highlighted and a state-of-the-art blueprint for conducting high-quality in-depth N- and O-glycoproteomic analyses is provided. Further, a modified and improved version of cotton hydrophilic interaction liquid chromatography-based solid phase extraction for glycopeptide enrichment is described. For the unambiguous identification of N-glycopeptides, the use of a conserved yet, rarely employed-fragmentation signature [Mpeptide+H+0,2X GlcNAc]+ is proposed. It is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, the use of the relative abundance of oxonium ions to retrieve glycan structure information, for example, differentiation of hybrid- and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc, is systematically and comprehensively evaluated. The findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics.

Conflict of Interest

The authors declare no conflict of interest.