Journal
PROTEOMICS
Volume 18, Issue 24, Pages -Publisher
WILEY
DOI: 10.1002/pmic.201800282
Keywords
fibrinogen; glycoproteomics; higher energy collision dissociation; hydrophilic interaction liquid chromatography; IgG; lactotransferrin; mass spectrometry; N-glycosylation; O-glycosylation; oxonium ions; proteinase K; ribonuclease B
Funding
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under the project The concert of dolichol-based glycosylation: from molecules to disease models [FOR2509]
- European Union (EC) [721815]
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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 [M-peptide+H+X-0,X-2 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.
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