Improved fractionation of sialylated glycopeptides by pellicular anion-exchange chromatography.
The glycoprotein bovine fetuin was treated with trypsin and the Asn-81 tryptic glycopeptide was purified (90% pure by Edman sequencing) by reversed-phase chromatography (RP-HPLC). The Asn-81 glycopeptide, which eluted as a single peak by RP-HPLC, was separable into five peaks on the NucleoPac PA100 column, a pellicular anion-exchange column. Each of the five Asn-81 glycopeptide peaks was shown to contain N-linked oligosaccharides by treatment of each peak with peptide N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) and subsequent oligosaccharide analysis by high-pH anion-exchange chromatography with pulsed amperometric detection. High-pH anion-exchange chromatography-pulsed amperometric detection oligosaccharide analysis revealed that each peak contained a different population of sialylated N-linked oligosaccharides. Hence each peak contained a different group of glycopeptide glycoforms. It was observed that the longer the retention time of the Asn-81 glycopeptide peak on the anion-exchange column, the greater the oligosaccharide sialylation. Two glycopeptide peaks which differed in their distribution of disialylated oligosaccharides demonstrated that the glycopeptide separation was a result of something more than gross differences in sialic acid content. The two other N-linked tryptic glycopeptides of fetuin were also separated into multiple peaks on the NucleoPac PA100 column and these separations were shown to be due to differences in oligosaccharide sialylation. The separations of the three fetuin N-linked glycopeptides demonstrate that pellicular anion-exchange chromatography offers improved separation speed and resolution for the separation of sialylated glycopeptides.
Rohrer
J S
JS
Dionex Corporation, Sunnyvale, CA 94086.
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D006868:Hydrolysis; D009844:Oligosaccharides / Q000032:analysis; D012795:Sialoglycoproteins / Q000302:isolation & purification*; D014357:Trypsin; D000509:alpha-Fetoproteins / Q000302:isolation & purification
D009844:Oligosaccharides; D012795:Sialoglycoproteins; D000509:alpha-Fetoproteins; D014357:Trypsin