TY - JOUR A1 - Kasim, Mumtaz A1 - Griebel, Anja A1 - Sandig, Grit A1 - Höltzel, Robert A1 - Malhotra, Akshay A1 - Hinderlich, Stephan A1 - Sandig, Volker A1 - Müller, Barbara A1 - von Horsten, Hans Henning ED - Halper, Jaroslava ED - Luo, Liang T1 - Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow JF - Bioengineering N2 - 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. KW - Glykane KW - glycoproteins KW - glycan analytics KW - -glycans KW - Hofmann rearrangement KW - glycan sample preparation KW - bioprocessing KW - FACE fluorescence-assisted carbohydrate electrophoresis KW - Glykoproteine KW - Arbeitsablauf Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:523-17922 SN - 2306-5354 VL - 10 IS - 6 PB - MDPI ER - TY - JOUR A1 - Kasim, Mumtaz A1 - Griebel, Anja A1 - Sandig, Grit A1 - Höltzel, Robert A1 - Malhotra, Akshay A1 - Hinderlich, Stephan A1 - Sandig, Volker A1 - Müller, Barbara A1 - von Horsten, Hans Henning ED - Halper, Jaroslava ED - Luo, Liang T1 - Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow JF - Bioengineering N2 - 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. KW - Glykoproteine KW - glycoproteins KW - glycan analytics KW - -glycans KW - Hofmann rearrangement KW - glycan sample preparation KW - bioprocessing KW - FACE fluorescence-assisted carbohydrate electrophoresis KW - Glykane KW - Bioverfahrenstechnik KW - Elektrophorese Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:523-17138 SN - 2306-5354 VL - 10 IS - 6 SP - 1 EP - 13 PB - MDPI ER - TY - JOUR A1 - Kasim, Mumtaz A1 - Schulz, Malissa A1 - Griebel, Anja A1 - Malhotra, Akshay A1 - Müller, Barbara A1 - von Horsten, Hans Henning T1 - Release of protein N-glycans by effectors of a Hofmann carboxamide rearrangement JF - Frontiers in Molecular Biosciences N2 - Background: Chemical methods for glycan release have gained traction because of their cost efficiency, accelerated reaction time and ability to release glycans not amenable to enzymatic cleavage. Oxidative chemical glycan release via hypochlorite treatment has been shown to be a convenient and efficient method that yields N-glycans similar to classical PNGase F digestion. We observed that the initial steps of the suggested mechanism for the oxidative release of glycans from glycoproteins by hypohalites showed similarities to the initiating steps of the classical Hofmann rearrangement of carboxamides. Therefore, we investigated the ability of different stable effectors of a Hofmann-type carboxamide rearrangement to efficiently and selectively release N-glycans from glycoproteins. Methods: Released glycans obtained from different experimental chemical release approaches were analyzed by HILIC-FLD, BHZ-FACE and ESI-MS and evaluated with respect to electrophoretic mobility, retention time and integrated peak area for resolved glycans. Results: We show that the known Hoffmann catalysts 1,3-dichloro-5,5-dimethylhydantoin, the hypervalent organoiodine (III) compound diacetoxy-iodobenzene as well as in-situ hypobromite generation using Oxone® and potassium bromide are all capable of releasing protein-bound N-glycans in good yield. Among the compounds investigated, diacetoxy-iodobenzene was capable of releasing glycans in the absence of alkali. Detailed investigations of the bromide/Oxone® method revealed a dependence of N-glycan release efficiency from the temporal order of bromide addition to the reaction mix as well as from a molar excess of bromide over Oxone®. Conclusions. These findings suggest that the oxidative release of N-glycans occurs via the initiating steps of a Hofmann carboxamide rearrangement. Hypervalent organoiodine compounds hold the promise of releasing glycans in the absence of alkali. The in-situ generation of hypobromite by bromide/Oxone® produces a consistent defined amount of reagent for rapid N-glycan release for both analytical and preparative purposes. KW - Biologika KW - glycan analytics KW - N-glycans KW - hofmann rearrangement KW - protein glycosylation KW - oligosaccharides KW - biopharmaceuticals Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:523-16082 SN - 2296-889X VL - 9 SP - 01 EP - 17 PB - Frontiers Media S.A. ER -