Rapid analysis of membrane glycopeptides by gel permeation high-performance liquid chromatography.
A rapid procedure for the analysis of glycopeptides has been developed using gel permeation high-performance liquid chromatography (HPLC). Glycopeptides derived by exhaustive pronase digestion of glycoproteins from radiolabeled human tumor and normal cell lines were chromatographed on DuPont GF-250 and GF-450 gel permeation columns in buffers containing non-ionic detergents. Effective separations of glycopeptides ranging in molecular mass from less than 600 daltons to more than 20,000 daltons, equivalent to the separation range of Sephadex G50 chromatography, were achieved in 7 min. The separations were dependent upon the use of an isocratic mobile phase, that contained a low-ionic-strength Tris buffer and Nonidet P-40 or Triton X-100. The mobilities of protein standards indicated the occurrence of a biphasic elution system, which favored the separation of species with molecular masses below 20,000 daltons. Glycopeptides isolated by this method could be applied directly to lectin or ion-exchange columns or could be digested with neuraminidase, endo H or other enzymes without further treatment. Removal of sialic acid from the glycopeptides caused a dramatic increase in retention time. Using this method, glycopeptides could be isolated rapidly and in high yield. The ease, speed and reproducibility of the separations and compatibility of the solvent systems with affinity or ion-exchange chromatography techniques make this gel permeation HPLC method an ideal initial step in the purification of glycopeptides.
Fuhrer
J P
JP
Human Tumor Marker Laboratory, Hipple Cancer Research Center, Dayton, OH 45439.
D000230:Adenocarcinoma; D001940:Breast; D001943:Breast Neoplasms; D002460:Cell Line; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003110:Colonic Neoplasms; D005260:Female; D005944:Glucosamine / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D008358:Mannose / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification*; D009439:Neuraminidase
D006020:Glycopeptides; D008562:Membrane Glycoproteins; D009439:Neuraminidase; D005944:Glucosamine; D008358:Mannose