Russian Journal of Bioorganic Chemistry, Vol. 26, No. 1, 2000, š . 45
The Degradation of Glycoproteins with Lithium Borohydride: Isolation and Analysis of O -Glycopeptides with Reduced C -Terminal Amino Acid Residue
N. P. Arbatsky*, 1 L. M. Likhosherstov*, M. V. Serebryakova**, O. S. Brusov**, V. N. Shibaev*, V. A. Derevitskaya*, † and N. K. Kochetkov*
*Zelinskii Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 117334 Russia, **National Mental Health Research Center, Russian Academy of Medical Sciences, Zagorodnoe sh. 2, korp. 2, Moscow , 113152 Russia
Abstract: By the example of fetuin and a blood-group-specific mucin from porcine stomach, we showed that, under conditions of reductive degradation of glycoproteins with LiBH4–LiOH in 70% aqueous tert-butyl alcohol, the reduction and cleavage of amide bonds occur much faster than the simultaneous -elimination of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are the O-glycosylated derivatives of 2-aminopropane-1,3-diol and 2-aminobutane-1,3-diol (the products of reduction of glycosylated Ser and Thr) and the glycopeptides containing 24 amino acid residues with reduced C-terminal amino acid. Seventeen homogeneous O-glycopeptides were isolated from the fetuin degradation products by ion-exchange and reversed-phase HPLC. Their structures were determined by MALDI-TOF mass spectrometry and by analyses for amino acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation sites in O- and N,O-glycoproteins is discussed.
Key words: O-glycoproteins, O-glycosylation sites; O-glycopeptides, analysis, chromatography, MALDI-TOF mass spectrometry