Fractionation of ovalbumin glycopeptide AC-C by high-pressure liquid chromatography Determination of structure by 1H-NMR spectroscopy

Analyzing Protein Micro-Heterogeneity in Chicken Ovalbumin by High-Resolution Native Mass Spectrometry Exposes Qualitatively and Semi-Quantitatively 59 Proteoforms

“…Inspection of the presented glycans with composition numbers 1−23 (Table 1) confirmed the presence of oligomannose-type, hybrid-type with and without intersecting GlcNAc, and complex-type di-, tri-(2,4 or 2,6 branching), and tetra-antennary (2,4 and 2,6 branching) structures with intersecting GlcNAc. Inspection of the remaining composition numbers 24−45, primarily heavier in mass and more complex, accounting cumulatively for 4.1% of the total ion intensity in the unprocessed ovalbumin mass spectra, showed for the numbers 25,27,28,30,34,37, and 44 a fit with glycan structures, earlier reported for pigeon ovalbumin (Table 1). 20 In the latter study, the assigned structures have been estimated by FAB-MS and ESI-MS in combination with methylation analysis and three-dimensional HPLC/exoglycosidase digestions and are part of a series of N-glycans containing terminal Gal(α1−4)Gal epitopes.…”

“…It should be noted that in our analysis we did not observe any traces of ovomucoid (28 kDa) or ovotransferrin (77 kDa), glycoproteins which should easily be mass-resolved from ovalbumin. For the majority of the 45 compositions in terms of Sia x Hex y HexNAc z , as presented in Table 1, we have shown the translation into real glycan structures based on Neu5Ac, Gal, Man, and GlcNAc, as available from the literature, in 18, and 23), [24][25][26][27]36 NMR spectroscopy (composition numbers 1, 2, 4, 6−8, 10−12, 14−16, and 18), 29,31,32,34,35,39,42 twodimensional (2D) high-performance liquid chromatography (HPLC) in combination with exoglycosidase digestions (composition numbers 2, 4, 6, 7, 8, 10, 11, 14, and 18), 37 MALDI-TOF MS in combination with exoglycosidase digestions, including NMR and enzymatic data of earlier reports (composition numbers 1−12, 14−16, 18, 19, and 22), 47 and detailed MS fragmentations studies (composition numbers 5, 6, and 10). 52 The references included in Table 1 and Figure S-2 of the Supporting Information refer to these original studies.…”

“…Over the past decades, the N-glycosylation of chicken ovalbumin has been examined by a plethora of analytical techniques, making it already, before this study, one of the best-characterized glycoproteins. − In fact, the glycoprotein has been quite often selected as a model compound in the development of new strategies for the structural analysis of N-glycosylation patterns, making use of chemically or enzymatically released carbohydrate chains from denatured material. It should be noted that in most studies commercially available ovalbumin preparations have been used instead of highly purified samples.…”

“…The symbolic notation of the N-glycan structures and linkages are explained in Supporting Information, Figure S-3. Most of the assigned structures are based on highly detailed previous studies on chicken ovalbumin using methylation analysis in combination with liquid chromatography/exoglycosidase digestions (composition numbers 1, 2, 4, 8, 10, 11, 14, 18, and 23), − , NMR spectroscopy (composition numbers 1, 2, 4, 6–8, 10–12, 14–16, and 18), ,,,,,, two-dimensional (2D) high-performance liquid chromatography (HPLC) in combination with exoglycosidase digestions (composition numbers 2, 4, 6, 7, 8, 10, 11, 14, and 18), MALDI-TOF MS in combination with exoglycosidase digestions, including NMR and enzymatic data of earlier reports (composition numbers 1–12, 14–16, 18, 19, and 22), and detailed MS fragmentations studies (composition numbers 5, 6, and 10) . The references included in Table and Figure S-2 of the Supporting Information refer to these original studies.…”