Hydrogen deuterium exchange reveals changes to protein dynamics of recombinant human erythropoietin upon N- and O- desialylation

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“… 548 − 551 In other glycoproteins, the only observed changes were increased dynamics through the protein upon glycan removal. 541 , 552 Some studies observed only very minor changes in HDX kinetics despite known effects on overall thermal stability upon glycan removal. 553 The available studies have thus far revealed a complex and very context-dependent relationship between glycosylation and structural dynamics.…”

“…Even with limited sequence coverage, HDX-MS has been a reliable tool for mapping binding interfaces and tracking structural changes upon protein–protein and protein–ligand interactions involving serum glycoproteins ,,− and receptors. ,,, Several studies have also used HDX-MS to assess the influence of glycosylation itself on the overall structure of serum glycoproteins with some interesting variability that reveals a complex relationship between a protein’s glycosylation state and its conformational dynamics. In some systems, HDX-MS showed mixed effects with some areas becoming stabilized and other regions showing increased accessibility upon glycan removal. − In other glycoproteins, the only observed changes were increased dynamics through the protein upon glycan removal. , Some studies observed only very minor changes in HDX kinetics despite known effects on overall thermal stability upon glycan removal . The available studies have thus far revealed a complex and very context-dependent relationship between glycosylation and structural dynamics.…”

Impact of Glycosylation on the Comparability of the Higher-Order Structures in Idursulfase by Hydrogen–Deuterium Exchange Mass Spectrometry

“…The changes in the N221/N225 region, including peptides adjacent in-space to the modified glycans, imply a synergistic change in the protein induced by the modification of both negatively charged terminal motifs. Previously, glycan related conformational change has also been reported for erythropoietin following desialylation …”

“…Previously, glycan related conformational change has also been reported for erythropoietin following desialylation. 25 Other analytical tools, such as differential scanning calorimetry (DSC), have been evaluated and are less informative (Figure S-7 and Table S-2). The differences observed by DSC as related to thermal stability are not as easily differentiated as compared to HDX-MS and do not provide any site-specific structural information as does HDX-MS. Other biophysical techniques such as circular dichroism, fluorescence spectroscopy, and so on are expected to provide similarly less information than HDX-MS for these modulated samples.…”