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

• Published in: Analytical chemistry (Washington) 2022-07, Vol.94 (27), p.9863-9871
• Main Authors: Gramlich, Marius, Maier, Sandra, Kaiser, Philipp D., Traenkle, Bjoern, Wagner, Teresa R., Voglmeir, Josef, Stoll, Dieter, Rothbauer, Ulrich, Zeck, Anne
• Format: Article
• Language: English
• Subjects: Chemistry; Deglycosylation; Deuterium; Epitope mapping; Glycan; Glycosylation; Hydrogen-deuterium exchange; Liquid chromatography; Mass spectrometry; Mass spectroscopy; N-glycanase; Nanobodies; Nucleotide sequence; Peptides; pH effects; Phosphine; Phosphines; Proteins; Scientific imaging; Spectroscopy; Substrate specificity; Substrates; Urea; Workflow
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.2c01748

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).