Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis

Holland, J. W., Deeth, H. C. and Alewood, P. F. (2005) Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis. Proteomics, 5 4: 990-1002.


Author Holland, J. W.
Deeth, H. C.
Alewood, P. F.
Title Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis
Journal name Proteomics  (ERA 2012 Listed)    (ERA 2010 Rank A)   Check publisher's open access policy
Publication date 2005
Sub-type Article
DOI 10.1002/pmic.200401098
Volume number 5
Issue number 4
ISSN 1615-9853
Start page 990
End page 1002
Total pages 13
Place of publication Weinheim
Publisher Wiley-VCH Verlag
Collection year 2005
Language eng
Subject C1
290104 Other Food Sciences
670105 Dairy products
Abstract The ability of two-dimensional gel electrophoresis (2-DE) to separate glycoproteins was exploited to separate distinct glycoforms of kappa-casein that differed only in the number of O-glycans that were attached. To determine where the glycans were attached, the individual glycoforms were digested in-gel with pepsin and the released glycopeptides were identified from characteristic sugar ions in the tandem mass spectrometry (MS) spectra. The O-glycosylation sites were identified by tandem MS after replacement of the glycans with ammonia/aminoethanethiol. The results showed that glycans were not randomly distributed among the five potential glycosylation sites in kappa-casein. Rather, glycosylation of the monoglycoform could only be detected at a single site, T-152. Similarly the diglycoform appeared to be modified exclusively at T-152 and T-163, while the triglycoform was modified at T-152, T-163 and T-154. While low levels of glycosylation at other sites cannot be excluded the hierarchy of site occupation between glycoforms was clearly evident and argues for an ordered addition of glycans to the protein. Since all five potential O-glycosylation sites can be glycosylated in vivo, it would appear that certain sites remain latent until other sites are occupied. The determination of glycosylation site occupancy in individual glycoforms separated by 2-DE revealed a distinct pattern of in vivo glycosylation that has not been recognized previously.
Keyword Biochemical Research Methods
Biochemistry & Molecular Biology
Casein
Glycoprotein
Glycosylation
Milk Protein
Post-translational Modification
Polypeptide N-acetylgalactosaminyltransferases
Mass-spectrometric Determination
Udp-galnac
K-casein
Carbohydrate Portions
Picomolar Sensitivity
Liquid-chromatography
Linked Glycosylation
Mapping Sites
Sugar Part
Q-Index Code C1

 
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