MASS-SPECTROMETRIC ANALYSIS OF INTEGRAL MEMBRANE-PROTEINS - APPLICATION TO COMPLETE MAPPING OF BACTERIORHODOPSINS AND RHODOPSIN

Integral membrane proteins have not been readily amenable to the gener al methods developed for mass spectrometric (or internal Edman degrada tion) analysis of soluble proteins. We present here a sample preparati on method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectro metry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyano gen bromide cleavage fragments of integral membrane proteins. This met hod has been applied to wild type (WT) bacteriorhodopsin (bR), cystein e containing mutants of bR, and the prototypical G-protein coupled rec eptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the pr otein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are s eparated by reversed phase HPLC using an isopropanol/acetonitrile/aque ous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of singl e amino acid fragments and the glycosylated fragment of Rh, which is o bservable by matrix assisted laser desorption ionization (MALDI)-MS, t his system permits analysis of the entire protein in a single HPLC run . This methodology will enable pursuit of chemical modification and cr osslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral memb rane proteins.