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Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H + , which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H + retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H + allowed the extraction of HDX data from all five glycosylated regions of the serpin α 1 -antichymotrypsin. We demonstrate that PNGase A and PNGase H + are capable of similar deglycosylation performance during HDX-MS analysis of α 1 -antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H + provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H + for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.

journal of the american society for mass spectrometry

Deglycosylation by the Acidic Glycosidase PNGase H<sup>+</sup> Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry

Deglycosylation by the Acidic Glycosidase PNGase H+ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry

Deglycosylation by the Acidic Glycosidase PNGase H⁺ Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry