Online Deglycosylation

Dr. Anne Zeck
Group Leader Bioanalytics

+49 7121 51530-811

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Use of a new enzyme for online deglycosylation in HDX-MS workflow

Protein-protein interaction sites or protein conformational changes can be performed by hydrogen-deuterium exchange (HDX), pepsin digestion and LC-MS analysis in medium sample throughput and with comparatively low amounts of material. The method resolution is highly dependent on the sequence coverage obtained by enzymatic digestion and LC-MS analysis. Protein glycosylation leads to gaps in the sequence coverage, as it is very heterogenous and therefore results in drastic reductions in detection sensitivity for single glycopeptide species.

We are working on implementing on-line deglycosylation into the HDX-MS workflow by using a new peptide N-glycanase (PNGase Rc) that is active under HDX quenching conditions (acidic pH and low temperature).

Publications

• Gramlich M et al. A Novel PNGase Rc for improved Protein N-Deglycosylation in Bioanalytics and Hydrogen-Deuterium Exchange coupled cith Mass Spectrometry Epitope Mapping under Challenging Conditions. Anal Chem. 2022 Jul 12;94(27):9863-9871.
  doi: 10.1021/acs.analchem.2c01748. Epub 2022 Jun 24. PMID: 35749695.