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.
Dionex Corporation, Sunnyvale, CA 94088-3603.
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D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A; D004076:Digoxigenin; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006020:Glycopeptides / Q000032:analysis*; D006868:Hydrolysis; D013058:Mass Spectrometry; D008666:Metalloendopeptidases; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010455:Peptides / Q000302:isolation & purification; D011134:Polysaccharides / Q000302:isolation & purification; D012260:Ribonucleases / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D006020:Glycopeptides; D010455:Peptides; D011134:Polysaccharides; D003208:Concanavalin A; D012260:Ribonucleases; C029600:ribonuclease B; D010450:Endopeptidases; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D004076:Digoxigenin