Sensitive blotting assay for the detection of glycopeptides in peptide maps

A dot blotting assay using digoxigenin hydrazide (Glycan detection kit, Boehringer Mannheim Biochemicals) was used to screen an endoproteinase Lys-C peptide map of ribonuclease B for the presence of glycopeptides. The carbohydrate content of the identified glycopeptide fraction was then further characterized by monosaccharide analysis using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). The tandem use of a hydrazide dot blotting technique to screen peptide maps for glycopeptides and subsequent use of HPAE-PAD to identify the monosaccharide composition of glycopeptide hydrolyzates proved to be a quick, sensitive and reliable method for identifying glycopeptides and analyzing their glycan composition without derivatization of the carbohydrate.