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

Autor: Udo Reichl, Robert Kottler, Alexander Pralow, Erdmann Rapp, René Hennig, Marcus Pioch, Marcus Hoffmann
Rok vydání: 2018
Předmět:
Zdroj: Proteomics
ISSN: 1615-9853
Popis: 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+0,2 X 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.
Databáze: OpenAIRE
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