A Novel PNGase Rc for Improved Protein N‑Deglycosylation in Bioanalytics and Hydrogen–Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions

N-linked glycosylation is a ubiquitous
posttranslational modification
of proteins. While it plays an important role in the biological function
of proteins, it often poses a major challenge for their analytical
characterization. Currently available peptide N-glycanases
(PNGases) are often inefficient at deglycosylating proteins due to
sterically inaccessible N-glycosylation sites. This usually leads
to poor sequence coverage in bottom-up analysis using liquid chromatography
with tandem mass spectrometry and makes it impossible to obtain an
intact mass signal in top-down MS analysis. In addition, most PNGases
operate optimally only in the neutral to slightly acidic pH range
and are severely compromised in the presence of reducing and denaturing
substances, which limits their use for advanced bioanalysis based
on hydrogen–deuterium exchange in combination with mass spectrometry
(HDX-MS). Here, we present a novel peptide N-glycanase
from Rudaea cellulosilytica (PNGase
Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively
folded proteins. Our results show that PNGase Rc is functional even
under challenging, HDX quenching conditions (pH 2.5, 0 °C) and
in the presence of 0.4 M tris­(2-carboxyethyl)­phosphine, 4 M urea,
and 1 M guanidinium chloride. Most importantly, we successfully applied
the PNGase Rc in an HDX-MS workflow to determine the epitope of a
nanobody targeting the extracellular domain of human signal-regulating
protein alpha (SIRPα).