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 fragmentat...

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Bibliographic Details
Published in:Journal of Chromatography A 1986, Vol.359, p.403-412
Main Authors: Tempst, Paul, Woo, David D.-L., Templow, David B., Aebersold, Ruedi, Hood, Leroy E., Kent, Stephen B.H.
Format: Article
Language:English
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Summary: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 C 4, C 18 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 (C 4) 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.
ISSN:0021-9673
DOI:10.1016/0021-9673(86)80094-2