Microscale elucidation of an N-linked glycosylation site by comparative high-performance liquid chromatographic...

The identification of specific sites of post-translational modifications of polypeptides is an important component in understanding protein structure-function relationships. This report describes the application of specific enzymatic methods of N-linked oligosaccharide removal (endo H and endo F) to the rapid microscale identification of specific sites of N-linked glycosylation. Following deglycosylation the protein is subjected to proteolytic digestion and comparative high-performance liquid chromatographic peptide mapping with an unmodified protein digest. Peptides from which oligosaccharide(s) have been removed exhibit a measurable increase in retention time on reversed-phase high-performance liquid chromatography and can be clearly identified. Once identified, the peptide can be subjected to direct protein microsequence analysis to elucidate the specific site of glycosylation. As illustrated here, these methods are compatible with microscale protein purification by sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by electroelution and are applicable to the 100-pmol range.