REVERSE-PHASE CHROMATOGRAPHY ISOLATION AND MALDI MASS-SPECTROMETRY OFTHE ACETYLCHOLINE-RECEPTOR SUBUNITS

A procedure for purifying the Torpedo californica nicotinic acetylchol ine receptor subunits is proposed which involves preparative SDS-PAGE followed by reverse-phase HPLC on a C-4 column in an acetonitrile-isop ropanol system. By this method, the alpha-subunit can be completely se parated from the 43-kDa protein which migrates very close to it on SDS -PAGE, and the delta-subunit can be isolated free from the beta-subuni t of Na+,K+-ATPase comigrating with it on SDS-PAGE. The purity of all acetylcholine receptor subunits thus obtained was verified by Edman de gradation and MALDI mass-spectrometric analysis which could be perform ed quite easily on the HPLC-purified samples. In general, we observed a good correlation between the experimentally determined molecular mas ses and those calculated from the amino acid sequences and, when known , posttranslational modifications (glycosylation and phosphorylation) of individual receptor subunits. Transfer of the isolated receptor sub units into 1% octyl-beta-D-glucopyranoside generates samples suitable for functional studies and enzymatic proteolysis or deglycosylation. ( C) 1998 Academic Press.