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

Gerard Comamala, Jeppe B. Madsen, Josef Voglmeir, Ya Min Du, Pernille F. Jensen, Eva C. Østerlund, Morten B. Trelle, Thomas J.D. Jørgensen*, Kasper D. Rand*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

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 α1-antichymotrypsin. We demonstrate that PNGase A and PNGase H+ are capable of similar deglycosylation performance during HDX-MS analysis of α1-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.

Original languageEnglish
JournalJournal of The American Society for Mass Spectrometry
Volume31
Issue number11
Pages (from-to)2305-2312
ISSN1044-0305
DOIs
Publication statusPublished - 2020

Keywords

  • deglycosylation
  • hydrogen/deuterium exchange mass spectrometry
  • mass spectrometry
  • PNGase

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