Title: Glycoproteomics of <b><i>N</i></b>‐glycosylation by in‐gel deglycosylation and matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry mapping: Application to congenital disorders of glycosylation
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.
Publication Year: 2005
Publication Date: 2005-06-28
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 61
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