Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins

• Published in: Protein science 1998-06, Vol.7 (6), p.1423-1430
• Main Authors: Whitelegge, Julian P., Gundersen, Cameron B., Faull, Kym F.
• Format: Article
• Language: English
• Subjects: Animals; Apoproteins - analysis; Bacteriorhodopsins - analysis; Cattle; Chloroplasts - ultrastructure; Chromatography, High Pressure Liquid; electrospray‐ionization; GTP-Binding Proteins - analysis; G‐protein coupled receptor; Halobacterium - chemistry; Intracellular Membranes - chemistry; intrinsic membrane protein; mass spectrometry; Mass Spectrometry - methods; Membrane Proteins - analysis; Photosynthetic Reaction Center Complex Proteins - analysis; post‐translational modifications; proteome; reverse‐phase HPLC; Rhodopsin - analysis; Spinacia oleracea - ultrastructure
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560070619

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray‐ionization mass spectrometry (ESI‐MS) of intact intrinsic membrane proteins purified using reverse‐phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the Dl and D2 reaction‐center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI‐MS study of a G‐protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post‐translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI‐MS experiments.