Characterization of integral membrane proteins using mass spectrometry : bacteriorhodopsin, rhodopsin, and neutrophil cytochrome b558
Year: 1998
Type: dissertation
Abstract: Electrospray ionization mass spectrometry (ESI MS) and matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) have been used extensively to characterize the structural properties of cytosolic proteins.However, ESI and MALDI have had limited success in determining the accurate mass of integral membrane proteins, presumably due to interference from the detergents needed to solubilize them.Therefore, mass spectrometry has had limited applicability in structural studies of integral membrane proteins.The integral membrane protein bacteriorhodopsin (BR) was used as a model in the development of a method for determining the accurate mass of BR solubilized in the detergents cetyltrimethylammonium bromide (CTAB), octyl-β-glucoside (OG), and CHAPSO by ESI MS.Quality mass spectra were obtained by means of a phase-separation technique where BR was partitioned into a chloroform-rich layer after mixing BR solubilized in an aqueous detergent solution with a chloroform:methanol:water mixture.Tryptic digestion of the G protein-coupled receptor rhodopsin was used as a test case for mapping the transmembrane peptides of an integral membrane protein using MALDI MS.Affinity purified rhodopsin solubilized in OG was digested with trypsin and the resulting proteolytic fragments were separated using a modified reverse-phase high performance liquid chromatography technique.Detergent was incorporated throughout the procedure.Transmembrane tryptic peptides were identified by accurate mass and confirmed by Edman microsequencing.The heterodimeric integral membrane protein cytochrome b558 from human neutrophils is the central component of the microbicidal NADPH-oxidase enzyme system.The protein shuttles electrons across the lipid bilayer via hemes located within the hydrophobic regions of the molecule to generate superoxide anion.Little is known about the tertiary structure of the protein, including the heme binding regions.Methods were developed for isolating fragments of purified cytochrome b558 that retained heme absorbance spectra after partial digestion with either trypsin or endoprotease Glu-C (V8) for analysis by SDS-PAGE, Western Blotting, and MALDI MS.Evidence from these analyses suggested that a region of the small subunit containing histidine 94 and regions of the large subunit of cytochrome b558 is involved in binding one of the hemes.
Author: David R. Barnidge
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2433947180