Determination of the structure of four glycopeptides from hen ovalbumin using 360-MHz proton magnetic resonance

Abstract

Four glycopeptides have been purified by Dowex and Bio-Gel P2 chromatography from Pronase digests of hen ovalbumin. The high-resolution proton magnetic resonance spectra of these glycopeptides and various products of their enzymatic digestion have been obtained at 360 MHz. By use of information derived from the spectra of a number of model compounds, an unambiguous assignment of all C1-H and Man C2-H resonances in the spectra can be made. On this basis structures are proposed for the four glycopeptides which are identical with those structures previously deduced from destructive chemical methods.

Separation of Neutral Glycoasparagines According to Their Content ofcisDiol Groups

“…1H-NMR data for the major fraction thus purified (denoted as GP-I) are summarized in Table II, these data being in good agreement with those reported by Carver et al 10 ) and van Halbeek et al 7 ) for GP-I and its derivative. The minor peaks observed in the borate chromatogram contained insufficient amount for a structural analysis.…”

Minimizing cationization effects in the analysis of complex mixtures of oligosaccharides

“…19 Using the example of the three major glycans from ovalbumin, Table 2 attempts to demonstrate the ease of fingerprinting the distribution of molecular weights using lithium chloride in MALDI mass spectrometric analysis of complex glycan mixtures. Some of the complex structures reported [18][19][20] for the oligosaccharides in ovalbumin together with the expected molecular weights as the 2-AMAC derivatives are shown in Fig. 4.…”

Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

“…3), pooled glyco voided volume were desalted on Bio-Gel P2 an samples were subsequently dissolved in 1 ml applied to L-PHA-agarose, and eluted with PBS constants of the C-1 protons from a-linked mannose residues were -2 Hz, and those from 3-linked GIcNAc and galactose residues were -7 to 8 Hz. Assignments are based on previous spectra obtained by this laboratory (25) as well as on published data from several laboratories which describe the spectral properties of identical and related compounds (2,3,27).…”

“…The LeclA phenotype correlates with reduced processing of Man5GlcNAc2Asn to complex carbohydrates and is appar-Man5GlcNAc2Asn as described (12). Purity was determined by 1H nuclear magnetic resonance (NMR) spectroscopy (3). D20 (99.8 and 99.996%) was from Stohler, Waltham, Mass.…”

“…The glycopeptides from VSV grown in CHO cells (CHO/VSV) were prepared in this laboratory, and their structures were determined previously by 'H NMR spectroscopy (25). The Man5GlcNAc2Asn glycopeptide was prepared from ovalbumin by the method of Huang et al (12), and its structure was determined by 'H NMR spectroscopy (3). lodination was achieved by mixing 10 to 100 ,ug of dried glycopeptide with 1 mCi of 125I-labeled Bolton Hunter reagent as described previously (1).…”