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

• Published in: Journal of the American Society for Mass Spectrometry Vol. 31; no. 11; pp. 2305 - 2312
• Main Authors: Comamala, Gerard, Madsen, Jeppe B, Voglmeir, Josef, Du, Ya-Min, Jensen, Pernille F, Østerlund, Eva C, Trelle, Morten B, Jørgensen, Thomas J. D, Rand, Kasper D
• Format: Journal Article
• Language: English
• Published: American Chemical Society 04.11.2020
• Subjects: mass spectrometry; deglycosylation; PNGase; hydrogen/deuterium exchange mass spectrometry
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1021/jasms.0c00258

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.