Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS
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Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS. / Lambert, Thomas; Gramlich, Marius; Stutzke, Luisa; Smith, Luke; Deng, Dingyu; Kaiser, Philipp D; Rothbauer, Ulrich; Benesch, Justin L P; Wagner, Cornelia; Koenig, Maximiliane; Pompach, Petr; Novak, Petr; Zeck, Anne; Rand, Kasper D.
In: Journal of the American Society for Mass Spectrometry, Vol. 34, No. 11, 2023, p. 2556–2566.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Development of a PNGase Rc Column for Online Deglycosylation of Complex Glycoproteins during HDX-MS
AU - Lambert, Thomas
AU - Gramlich, Marius
AU - Stutzke, Luisa
AU - Smith, Luke
AU - Deng, Dingyu
AU - Kaiser, Philipp D
AU - Rothbauer, Ulrich
AU - Benesch, Justin L P
AU - Wagner, Cornelia
AU - Koenig, Maximiliane
AU - Pompach, Petr
AU - Novak, Petr
AU - Zeck, Anne
AU - Rand, Kasper D
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
U2 - 10.1021/jasms.3c00268
DO - 10.1021/jasms.3c00268
M3 - Journal article
C2 - 37756257
VL - 34
SP - 2556
EP - 2566
JO - Journal of The American Society for Mass Spectrometry
JF - Journal of The American Society for Mass Spectrometry
SN - 1044-0305
IS - 11
ER -
ID: 368803480