Method for the detection of glycopeptides at the picomole level in HPLC peptide maps.
Glycopeptide-containing fractions in HPLC peptide maps can be detected by a simple application of the microtiter plate-bound streptavidin-biotinylated glycopeptide-lectin method (M.-C. Shao, 1992, Anal. Biochem., 205, 77-82). To illustrate this application, the glycoproteins, ovalbumin and asialofetuin, reduced and S-alkylated with vinylpyridine, were digested with trypsin-L-1-p-tosylamino-2-phenylethylchloromethyl ketone and the tryptic peptides were fractionated by reverse-phase HPLC, monitoring for absorbance at 230 nm. Aliquots of the HPLC fractions (typically 0.2-0.5% of the total volume) were biotinylated and complexed with streptavidin in the wells of a microtiter plate, allowing the streptavidin-glycopeptide complex to adhere to the plate. Suitable lectins, such as concanavalin A, Datura stramonium agglutinin, and peanut agglutinin, all of which had been coupled to horse radish peroxidase, were added, and after thorough washing, only the wells containing streptavidin-bound glycopeptides retained the complementary lectin and gave a positive peroxidase reaction. Less than 1 pmol of glycopeptide can be detected. The demonstration that the glycopeptide detection could be inhibited either by addition of an excess of the appropriate sugar inhibitor to the different lectins or by digestion of the biotinylated glycopeptides with N-glycosidase F or O-glycosidase shows that the glycopeptide-lectin interaction is the basis for the reaction.
D000595:Amino Acid Sequence; D001212:Asialoglycoproteins / Q000032:analysis; D001710:Biotin; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D060748:Fetuins; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D037102:Lectins; D008832:Microchemistry; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping / Q000379:methods*; D010544:Peroxidases; D011134:Polysaccharides / Q000032:analysis; D012680:Sensitivity and Specificity; D000509:alpha-Fetoproteins / Q000032:analysis
D001212:Asialoglycoproteins; D060748:Fetuins; D006020:Glycopeptides; D037102:Lectins; D010446:Peptide Fragments; D011134:Polysaccharides; D000509:alpha-Fetoproteins; C017029:asialofetuin; D001710:Biotin; D010047:Ovalbumin; D010544:Peroxidases