Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins, and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

BioCev Institute of Biotechnology of the CAS 252 50 Prumyslova Czech Republic

BioCeV Institute of Microbiology of the CAS 142 20 Prumyslova Czech Republic

Department of Pharmacy University of Copenhagen 2100 Copenhagen Denmark

NMI Natural and Medical Sciences Institute at the University of Tübingen 72770 Reutlingen Germany

Pharmaceutical Biotechnology Eberhard Karls University 72074 Tübingen Germany

Physical and Theoretical Chemistry Laboratory Department of Chemistry University of Oxford OX1 3QZ Oxford England

Roche Pharma Research and Early Development Large Molecule Research Roche Innovation Center Munich 82377 Penzberg Germany

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Bakheet T. M.; Doig A. J. Properties and Identification of Human Protein Drug Targets. Bioinformatics 2009, 25 (4), 451–457. 10.1093/bioinformatics/btp002. PubMed DOI

Shental-Bechor D.; Levy Y. Folding of Glycoproteins: Toward Understanding the Biophysics of the Glycosylation Code. Curr. Opin. Struct. Biol. 2009, 19 (5), 524–533. 10.1016/j.sbi.2009.07.002. PubMed DOI

Varki A. Biological Roles of Glycans. Glycobiology 2017, 27 (1), 3.10.1093/glycob/cww086. PubMed DOI PMC

Varki A. Evolutionary Forces Shaping the Golgi Glycosylation Machinery: Why Cell Surface Glycans Are Universal to Living Cells. Cold Spring Harb. Perspect. Biol. 2011, 3 (6), a005462.10.1101/cshperspect.a005462. PubMed DOI PMC

Trabjerg E.; Nazari Z. E.; Rand K. D. Conformational Analysis of Complex Protein States by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS): Challenges and Emerging Solutions. TrAC - Trends Anal. Chem. 2018, 106, 125–138. 10.1016/j.trac.2018.06.008. DOI

Harrison R. A.; Engen J. R. Conformational Insight into Multi-Protein Signaling Assemblies by Hydrogen–Deuterium Exchange Mass Spectrometry. Curr. Opin. Struct. Biol. 2016, 41, 187–193. 10.1016/j.sbi.2016.08.003. PubMed DOI PMC

Vadas O.; Burke J. E. Probing the Dynamic Regulation of Peripheral Membrane Proteins Using Hydrogen Deuterium Exchange-MS (HDX-MS). Biochem. Soc. Trans. 2015, 43, 773–786. 10.1042/BST20150065. PubMed DOI

Masson G. R.; Burke J. E.; Ahn N. G.; Anand G. S.; Borchers C.; Brier S.; Bou-Assaf G. M.; Engen J. R.; Englander S. W.; Faber J.; Garlish R.; Griffin P. R.; Gross M. L.; Guttman M.; Hamuro Y.; Heck A. J. R.; Houde D.; Iacob R. E.; Jorgensen T. J. D.; Kaltashov I. A.; Klinman J. P.; Konermann L.; Man P.; Mayne L.; Pascal B. D.; Reichmann D.; Skehel M.; Snijder J.; Strutzenberg T. S.; Underbakke E. S.; Wagner C.; Wales T. E.; Walters B. T.; Weis D. D.; Wilson D. J.; Wintrode P. L.; Zhang Z.; Zheng J.; Schriemer D. C.; Rand K. D. Recommendations for Performing, Interpreting and Reporting Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) Experiments. Nat. Methods 2019, 16 (7), 595–602. 10.1038/s41592-019-0459-y. PubMed DOI PMC

Jensen P. F.; Comamala G.; Trelle M. B.; Madsen J. B.; Jørgensen T. J. D.; Rand K. D. Removal of N-Linked Glycosylations at Acidic PH by PNGase a Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-linked Glycoproteins. Anal. Chem. 2016, 88 (24), 12479–12488. 10.1021/acs.analchem.6b03951. PubMed DOI

Comamala G.; Madsen J. B.; Voglmeir J.; Du Y.-M.; Jensen P. F.; Østerlund E. C.; Trelle M. B.; Jørgensen T. J. D.; Rand K. D. Deglycosylation by the Acidic Glycosidase PNGase H+Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2020, 31 (11), 2305–2312. 10.1021/jasms.0c00258. PubMed DOI

Guttman M.; Scian M.; Lee K. K. Tracking Hydrogen/Deuterium Exchange at Glycan Sites in Glycoproteins by Mass Spectrometry. Anal. Chem. 2011, 83 (19), 7492–7499. 10.1021/ac201729v. PubMed DOI PMC

Ford K. L.; Zeng W.; Heazlewood J. L.; Bacic A. Characterization of Protein N-Glycosylation by Tandem Mass Spectrometry Using Complementary Fragmentation Techniques. Front. Plant Sci. 2015, 6 (AUG), 147055.10.3389/fpls.2015.00674. PubMed DOI PMC

Mechref Y.Use of CID/ETD Mass Spectrometry to Analyze Glycopeptides. Curr. Protoc. Protein Sci. 2012, 68 ( (1), )10.1002/0471140864.ps1211s68. PubMed DOI PMC

Rand K. D.; Zehl M.; Jørgensen T. J. D. Measuring the Hydrogen/Deuterium Exchange of Proteins at High Spatial Resolution by Mass Spectrometry: Overcoming Gas-Phase Hydrogen/Deuterium Scrambling. Acc. Chem. Res. 2014, 47 (10), 3018–3027. 10.1021/ar500194w. PubMed DOI

Rand K. D.; Zehl M.; Jensen O. N.; Jørgensen T. J. D. Protein Hydrogen Exchange Measured at Single-Residue Resolution by Electron Transfer Dissociation Mass Spectrometry. Anal. Chem. 2009, 81 (14), 5577–5584. 10.1021/ac9008447. PubMed DOI

Comamala G.; Krogh C. C.; Nielsen V. S.; Kutter J. P.; Voglmeir J.; Rand K. D. Hydrogen/Deuterium Exchange Mass Spectrometry with Integrated Electrochemical Reduction and Microchip-Enabled Deglycosylation for Epitope Mapping of Heavily Glycosylated and Disulfide-Bonded Proteins. Anal. Chem. 2021, 93 (49), 16330–16340. 10.1021/acs.analchem.1c01728. PubMed DOI

Guo R. R.; Zhang T. C.; Lambert T. O. T.; Wang T.; Voglmeir J.; Rand K. D.; Liu L.. PNGase H+ Variant from Rudaea Cellulosilytica with Improved Deglycosylation Efficiency for Rapid Analysis of Eukaryotic N-Glycans and Hydrogen Deuterium Exchange Mass Spectrometry Analysis of Glycoproteins. Rapid Commun. Mass Spectrom. 2022, 36 ( (21), )10.1002/rcm.9376. PubMed DOI PMC

Wang T.; Cai Z. P.; Gu X. Q.; Ma H. Y.; Du Y. M.; Huang K.; Voglmeir J.; Liu L. Discovery and Characterization of a Novel Extremely Acidic Bacterial N-Glycanase with Combined Advantages of PNGase F and A. Biosci. Rep. 2014, 34 (6), 673–684. 10.1042/BSR20140148. PubMed DOI PMC

Gramlich M.; Maier S.; Kaiser P. D.; Traenkle B.; Wagner T. R.; Voglmeir J.; Stoll D.; Rothbauer U.; Zeck A. A Novel PNGase Rc for Improved Protein N-Deglycosylation in Bioanalytics and Hydrogen–Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions. Anal. Chem. 2022, 94, 9863.10.1021/acs.analchem.2c01748. PubMed DOI

Kurosky A.; Barnett D. R.; Lee T. H.; Touchstone B.; Hay R. E.; Arnott M. S.; Bowman B. H.; Fitch W. M. Covalent Structure of Human Haptoglobin: A Serine Protease Homolog. Proc. Natl. Acad. Sci. U. S. A. 1980, 77, 3388–3392. 10.1073/pnas.77.6.3388. PubMed DOI PMC

Guo R. R.; Comamala G.; Yang H. H.; Gramlich M.; Du Y. M.; Wang T.; Zeck A.; Rand K. D.; Liu L.; Voglmeir J.. Discovery of Highly Active Recombinant PNGase H+ Variants Through the Rational Exploration of Unstudied Acidobacterial Genomes. Front. Bioeng. Biotechnol. 2020, 810.3389/fbioe.2020.00741. PubMed DOI PMC

Kumai T.; Matsuda Y.; Ohkuri T.; Oikawa K.; Ishibashi K.; Aoki N.; Kimura S.; Harabuchi Y.; Celis E.; Kobayashi H. C-Met Is a Novel Tumor Associated Antigen for T-Cell Based Immunotherapy against NK/T Cell Lymphoma. Oncoimmunology 2015, 4 (2), e976077.10.4161/2162402X.2014.976077. PubMed DOI PMC

Mesteri I.; Schoppmann S. F.; Preusser M.; Birner P. Overexpression of CMET Is Associated with Signal Transducer and Activator of Transcription 3 Activation and Diminished Prognosis in Oesophageal Adenocarcinoma but Not in Squamous Cell Carcinoma. Eur. J. Cancer 2014, 50 (7), 1354–1360. 10.1016/j.ejca.2014.01.022. PubMed DOI

Feige M. J.; Hendershot L. M. Disulfide Bonds in ER Protein Folding and Homeostasis. Curr. Opin. Cell Biol. 2011, 23 (2), 167–175. 10.1016/j.ceb.2010.10.012. PubMed DOI PMC

Lambert T. O. T.; Gramlich M.; Stutzke L.; Smith L.; Deng D.; Kaiser P. D.; Benesch J.; Rothbauer U.; Vankova P.; Pompach P.; Novak P.; Koenig M.; Wagner C.; Zeck A.; Rand K. D.. Development of a PNGase Rc Column for Efficient Online Deglycosylation of Complex Glycoproteins during HDX-MS. WP 286, June 7th 2023, Proc. ASMS Conf. Mass Spectrom. Allied Top. Houston, Texas June 4-8th 2023 ASMS 2023.

Lambert T.; Gramlich M.; Stutzke L.; Smith L.; Deng D.; Kaiser P. D.; Rothbauer U.; Benesch J. L. P.; Wagner C.; Koenig M.; Pompach P.; Novak P.; Zeck A.; Rand K.. Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS. bioRxiv 2023, 2023.07.28.550801. PubMed PMC

O’Leary T. R.; Balasubramaniam D.; Hughes K.; Foster D.; Boyles J.; Coleman K.; Griffin P. R. Hydrogen-Deuterium Exchange Epitope Mapping of Glycosylated Epitopes Enabled by Online Immobilized Glycosidase. Anal. Chem. 2023, 95 (27), 10204.10.1021/acs.analchem.3c00374. PubMed DOI PMC

Structural Characterization of Monoclonal Antibodies and Epitope Mapping by FFAP Footprinting

Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS