Abstract |
A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate- polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N- glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N- glycans was compared. The most favourable results for native acidic N- glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N- glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented.
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Authors | Dijana Sagi, Petra Kienz, Jonas Denecke, Thorsten Marquardt, Jasna Peter-Katalinić |
Journal | Proteomics
(Proteomics)
Vol. 5
Issue 10
Pg. 2689-701
(Jul 2005)
ISSN: 1615-9853 [Print] Germany |
PMID | 15912511
(Publication Type: Journal Article)
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Chemical References |
- Glycoproteins
- Polysaccharides
- Sialic Acids
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Topics |
- Carbohydrate Conformation
- Carbohydrate Sequence
- Chromatography, Ion Exchange
- Electrophoresis, Polyacrylamide Gel
- Glycoproteins
(chemistry, isolation & purification, metabolism)
- Glycosylation
- Molecular Sequence Data
- Polysaccharides
(chemistry, isolation & purification)
- Proteomics
- Sialic Acids
(isolation & purification)
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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