Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins.

Abstract

C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identification of these mannosylated residues in proteins is rather tedious. In a previous paper, it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three different steps of gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptafluorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C((2))-mannosylated Trp could be quantitatively recovered and identified during the step of amino acid analysis. Analyses of different samples indicated that this type of glycosylation is absent in bacteria and yeasts.

Cite this paper

@article{Zanetta2004QuantitativeGC, title={Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins.}, author={Jean-Pierre Zanetta and Alexandre Pons and Colette Richet and Guillemette M Huet and Philippe Timmerman and Yves Leroy and Anne Bohin and J. P. Bohin and Pierre-Andr{\'e} Trinel and Daniel E. Poulain and Jan Hofsteenge}, journal={Analytical biochemistry}, year={2004}, volume={329 2}, pages={199-206} }