Mass Spectrometry Of Nicotinic Acetylcholine Receptors And Associated Proteins As Models For Complex Transmembrane Proteins
Studies were conducted to optimize matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI TOF MS) in analyzing the composition of nicotinic acetylcholine receptors (nAChR) from Torpedo californica electric tissue in their membrane-bound, detergent-solubilized, and affinity-purified states. Mass spectra obtained from nAChR-rich membrane fractions gave reasonably good representations of protein compositions indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of those same samples. Efficiency of extraction of nAChR from membranes was not markedly different for most detergents, but quality and signal size of mass spectra were clearly influenced by detergent composition and concentration, protein concentration, and MALDI matrix composition. The best spectra, allowing detection and accurate size determinations for samples containing as little as 10 fmol of pure nAChR, were obtained for samples solubilized in Triton X-100 and assayed by use of a sinapinic acid matrix. Although informative spectra could be obtained for nAChR affinity purified on Î±-cobratoxin (Naja naja siamensis) columns and extracted using sinapinic acid, superior spectra with much higher signal:noise were obtained if extraction media contained Triton X-100 or sodium dodecyl sulfate. nAChR subunit masses determined were similar regardless of the membrane-associated, detergent-solubilized, or affinity-purified state of the preparation. These studies illustrate how masses can be determined for nAChR subunits and for other protein components in Torpedo membrane preparations, such as RAPsyn and Na+-K+-ATPase Î± and Î² subunits. They also provide an underpinning for streamlined analysis of the composition of complex transmembrane proteins using MALDI TOF MS. Â© 2002 Elsevier Science (USA).
Digital Object Identifier (DOI)
Lukas, Ronald J.; Tubbs, Kemmons A.; Krivoshein, Arcadius V.; Bieber, Allan L.; and Nelson, Randall W., "Mass Spectrometry Of Nicotinic Acetylcholine Receptors And Associated Proteins As Models For Complex Transmembrane Proteins" (2002). Neurobiology. 245.