The degradation of glycoproteins with lithium borohydride: Isolation and analysis of O-glycopeptides with reduced C-terminal amino acid residue

By the example of fetuin and a blood-group-specific mucin from porcine stom ach, we showed that, under conditions of reductive degradation of glycoprot eins with LiBH4-LiOH in 70% aqueous tert-butyl alcohol, the reduction and c leavage of amide bonds occur much faster than the simultaneous p-eliminatio n of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are t he 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 gl ycopeptides containing 2-4 aminoacid 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 structu res were determined by MALDI-TOF mass spectrometry and by analyses for amin o acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation site s in O- and N,O-glycoproteins is discussed.