Chemistry

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 wer