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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F23%3A00581603" target="_blank" >RIV/86652036:_____/23:00581603 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/61388971:_____/23:00581603

Výsledek na webu

<a href="https://pubs.acs.org/doi/10.1021/jasms.3c00268" target="_blank" >https://pubs.acs.org/doi/10.1021/jasms.3c00268</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/jasms.3c00268" target="_blank" >10.1021/jasms.3c00268</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2023

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of the American Society for Mass Spectrometry

ISSN

1044-0305

e-ISSN

1879-1123

Svazek periodika

34

Číslo periodika v rámci svazku

11

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

11

Strana od-do

2556-2566

Kód UT WoS článku

001074666000001

EID výsledku v databázi Scopus

2-s2.0-85175202999