Naslov
Evaluation of different PNGase F enzymes in Immunoglobulin G and total plasma N-glycans characterization

Autori
Vilaj, Marija ; Trbojević-Akmačić, Irena ; Lauc, Gordan

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
BOOK OF ABSTRACTS / - , 2017, 113-113

Skup
12th Jenner Glycobiology and Medicine Symposium: Translational Glycobiology-From Bench to Bedside

Mjesto i datum
Dubrovnik, Hrvatska, 06.05.2017. - 09.05.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Podatak o recenziji nije dostupan

Ključne riječi
N-glycan ; Immunoglobulin G ; Plasma Glycoproteins ; PNGase F

Sažetak
Deglycosylation is one of the main steps in sample preparation for N-glycan analysis. Nglycosidase F (PNGase F) is an enzyme most widely used for releasing N-glycans from glycoproteins by incubation of deglycosylation mixture for a few hours or overnight at 37°C. The aim of this study was to compare different PNGase F enzymes (Rapid PNGase F and two recombinant versions from different producers) for deglycosylation of total plasma glycoproteins and different amounts of immunoglobulin G (IgG). Deglycosylation with Rapid PNGase F was done in corresponding Rapid PNGase F buffers and lasted for 10 minutes, while recombinant versions of PNGase F were tested in the same reaction conditions with 18 hour deglycosylation. Original deglycosylation protocols were modified to be compatible with in house in solution labeling with 2-aminobenzamide (2-AB), cleanup of 2-AB labeled N-glycans and subsequent ultra performance liquid chromatography (UPLC) analysis. Deglycosylation with different PNGase F enzymes resulted in different IgG/Plasma N-glycosylation hydrophilic interaction liquid chromatography (HILIC) UPLC profiles. Additionally, we observed that one recombinant version of PNGase F is more efficient in deglycosylation of complex N-glycans compared to Rapid version and recombinant version of PNGase F from a different producer. In terms of intensities and coefficient of variation (CV) values, all tested versions of PNGase F enzymes were very reproducible and on the similar level.

Izvorni jezik
Engleski

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