Glycoproteomics of N‐glycosylation by in‐gel deglycosylation and matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry mapping: Application to congenital disorders of glycosylation

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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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