Separation of Neutral Glycoasparagines According to Their Content ofcisDiol Groups

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

“…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.…”

“…It should be noted that in most studies commercially available ovalbumin preparations have been used instead of highly purified samples. In view of this, it has been reported that among the many assigned glycan structures in the literature, several structures do not originate from ovalbumin but from contaminating glycoproteins, such as ovomucoid, 39 , 47 , 58 − 63 ovotransferrin, 32 , 39 or riboflavin-binding protein. 47 , 64 , 65 Although for our research we have used commercial chicken ovalbumin (grade V), our data exclude such possibilities; as by analyzing the intact glycoprotein instead of the released glycans, we unambiguously link the observed glycan compositional data to the ovalbumin polypeptide backbone.…”

“…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.…”

Site-specific de- N -glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N -glycanase as a quality control system for newly synthesized proteins

“…Endo-␤-N-acetylglucosaminidase, which was previously identified in hen oviduct (31), was proposed to be a candidate for the enzyme responsible to deglycosylation of di-N-glycosylated OVA (18). However, this seems to be an unlikely possibility because no OVA-derived glycopeptide containing only GlcNAc residue on the Asn residue was detected (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). Wiseman et al (43) reported a variant form of OVA in which Asp residue replaced Asn-311, a second potential glycosylation site in genuine OVA.…”

Identification of two discrete peptide: N-glycanases inOryzias latipes during embryogenesis

“…Ovalbumin GlcNAc-peptide was prepared from the ovalbumin glycopeptides according to the method of Suzuki et al (1993). Glycoasparagine, Asn(Man 5 -GlcNAc 4 ) (GP-IIIA), was prepared from ovalbumin as described previously (Nomoto et al, 1992). Stem bromelain glycopeptide, Ala-Arg-Val-Pro-Arg-Asn-Asn(Fuc 0.86 Xyl 1.0 Man 2.6 -GlcNAc 2.0 )-Glu-Ser-Ser-Met, and its defucosylated and partially dexylosylated sample obtained by mild acid hydrolysis, Ala-ArgVal-Pro-Arg-Asn-Asn(Xyl 0.52 Man 2.6 GlcNAc 2.0 )-Glu-Ser-Ser-Met, were prepared as described previously (Suzuki et al, 1994a).…”