Bioengineering, Vol. 10, Pages 651: Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow

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Bioengineering, Vol. 10, Pages 651: Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow

Bioengineering doi: 10.3390/bioengineering10060651

Authors:
Mumtaz Kasim
Anja Griebel
Grit Sandig
Robert Höltzel
Akshay Malhotra
Stephan Hinderlich
Volker Sandig
Barbara Müller
Hans Henning von Horsten

Analytical methods fr direct quantitative N-glycan analysis require a sequence of sample preparation and clean-up steps that result in reduced glycan recovery. Therefore, we aimed to combine glycan release and labeling steps. Based on the hypothesis that the reaction mechanism for oxidative chemical glycan release comprises a stable glycan isocyanate intermediate, we investigated whether this could be exploited for the in-situ preparation of fluorescent glycan conjugates. ANTS-labeled N-glycans were derived from chicken ovalbumin via an in-situ chemical release/coupling approach and by standard Peptide-N-Glycosidase F (PNGase F) digestion/reductive amination. Synoptic fluorescence-assisted carbohydrate electrophoresis with UV detection (FACE-UV) analysis yielded matching patterns of fluorescent N-glycan bands in the expected electrophoretic mobility range between hexose units GU-5 and GU-11 of the standard. Anthranilamide (2-AB)-glycan conjugates prepared from a test glycoprotein carrying a predominant Core-F glycan gave single predominant peaks in hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) and electrospray ionization mass spectrometry (ESI-MS) spectra in agreement with sodiated triply charged Core-F-AB conjugates for both the standard and the in-situ coupling methods. The Core-F-AB conjugate prepared by the in-situ coupling approach had a slightly elevated retention time on HILIC-FLD and an ESI-MS m/z peak in line with a urea-bonded glycan-AB conjugate, with closed pyran ring structures on the glycan moiety. Glycan isocyanates intermittently formed during chemical glycan release, which could be utilized to prepare labeled glycan samples directly from glycoproteins and fluorescent dyes bearing a primary amine functional group.

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