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  • Title: Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins.
    Author: Jensen PF, Comamala G, Trelle MB, Madsen JB, Jørgensen TJ, Rand KD.
    Journal: Anal Chem; 2016 Dec 20; 88(24):12479-12488. PubMed ID: 28193043.
    Abstract:
    Protein glycosylation is the most frequent post-translational modification and is present on more than 50% of eukaryotic proteins. Glycosylation covers a wide subset of modifications involving many types of complex oligosaccharide structures, making structural analysis of glycoproteins and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX information can typically not be obtained from regions of the glycoprotein that contain the actual N-linked glycan as glycan heterogeneity combined with pepsin digestion yields a large diversity of peptic N-glycosylated peptides that can be difficult to detect. Here, we present a novel HDX-MS workflow for analysis of the conformational dynamics of N-linked glycoproteins that utilizes the enzyme PNGase A for deglycosylation of labeled peptic N-linked glycopeptides at HDX quench conditions, i.e., acidic pH and low temperature. PNGase A-based deglycosylation is thus performed after labeling (post-HDX) and the utility of this approach is demonstrated during analysis of the monoclonal antibody Trastuzumab for which it has been shown that the native conformational dynamics is dependent on the N-linked glycan. In summary, the HDX-MS workflow with integrated PNGase A deglycosylation enables analysis of the native HDX of protein regions containing N-linked glycan sites and should thus significantly improve our ability to study the conformational properties of glycoproteins.
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