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.
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.
Sagi
Dijana
D
Institute for Medical Physics and Biophysics, University of Münster, Münster, Germany.
Kienz
Petra
P
Denecke
Jonas
J
Marquardt
Thorsten
T
Peter-Katalinić
Jasna
J
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics; D012794:Sialic Acids / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006023:Glycoproteins; D011134:Polysaccharides; D012794:Sialic Acids