Microscale structure analysis of a high-molecular-weight, hydrophobic membrane glycoprotein fraction with platelet-derived growth factor-dependent kinase activity.
General methods for the study of the primary structure of picomole quantities of large, hydrophobic membrane glycoproteins with blocked amino-termini have been developed. Three techniques designed to be used in concert with each other are described: first, modified protein preparation and fragmentation techniques; secondly, a simple but very selective two-dimensional reversed-phase high-performance liquid chromatography system for the resolution of complex mixtures of small to medium-sized tryptic peptides on Vydac C4, C18 and diphenyl columns and thirdly, a two-dimensional separation method for large, denaturated (CNBr) polypeptide fragments by size-exclusion high-performance liquid chromatography, combined with either reversed-phase high-performance liquid chromatography (C4) or sodium dodecyl sulphate polyacrylamide gel electrophoresis in conjunction with electroblotting and autoradiography. These methods were applied to studies of the platelet-derived growth factor receptor. Starting with 500 pmoles of purified protein, a total of 232 amino acids were sequenced.
D000478:Alkylation; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D010084:Oxidation-Reduction; D010982:Platelet-Derived Growth Factor / Q000032:analysis*; D011494:Protein Kinases / Q000032:analysis*
D006023:Glycoproteins; D010982:Platelet-Derived Growth Factor; D011494:Protein Kinases