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

The unambiguous mass spectrometric identification and characterization of glycopeptides is crucial to elucidate the micro- and macroheterogeneity of glycoproteins. Here, we propose combining lower and stepped collisional energy fragmentation for the in-depth and site-specific analysis of N- and O-glycopeptides. Using a set of four representative and biopharmaceutically-relevant glycoproteins (IgG, fibrinogen, lactotransferrin, and ribonuclease B), we highlight the benefits and limitations of the developed workflow and provide a state-of-the-art blueprint for conducting high-quality in-depth N- and O-glycoproteomic analyses. Further, we describe a modified and improved version of cotton HILIC-based solid phase extraction for glycopeptide enrichment. For the unambiguous identification of N-glycopeptides we propose the use of a conserved fragmentation signature [Mpeptide +H+0,2 X GlcNAc]+ , that has rarely been employed in glycoproteomic analyses up to now. We show for the first time that this fragmentation signature can consistently be found across all N-glycopeptides, but not on O-glycopeptides. Moreover, we have systematically and comprehensively evaluated the use of the relative abundance of oxonium ions to retrieve glycan structure information, e.g. differentiation of hybrid- and high-mannose-type N-glycans or differentiation between antenna GlcNAc and bisecting GlcNAc. Our findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics. This article is protected by copyright. All rights reserved.