Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin

Protein Science : a Publication of the Protein Society
L E BallD R Knapp

Abstract

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization ...Continue Reading

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Citations

Feb 27, 2001·Mass Spectrometry Reviews·J Godovac-Zimmermann, L R Brown
Jul 20, 2007·Analytical and Bioanalytical Chemistry·Bernhard GranvoglLutz Andreas Eichacker
Mar 29, 2005·Journal of the American Society for Mass Spectrometry·Hongying ZhongLiang Li
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