Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry.
We demonstrate here that the hydrogen/deuterium solvent exchange (HDX) properties of the transmembrane fragment of the M2 protein of Influenza A (M2-TM) incorporated into lipid vesicles or detergent micelles can be studied with straightforward electrospray (ESI) and nanospray mass spectrometry (MS) configurations provided that key factors, including sample preparation techniques, are optimized. Small unilamellar vesicle preparations were obtained by solubilizing dimyristoyl phosphatidylcholine (DMPC) and the M2-TM peptide in aqueous solution with n-octyl-beta-D-glycopyranoside, followed by dialysis to remove the detergent. Electron microscopy experiments revealed that subsequent concentration by centrifugation introduced large multilamellar aggregates that were not compatible with ESI-MS. By contrast, a lyophilization-based concentration procedure, followed by thawing above the liquid crystal transition temperature of the lipid component, maintained the liposome size profile and yielded excellent ion fluxes in both ESI-MS and nano-ESI-MS. Using these methods the global HDX profile of M2-TM in aqueous DMPC vesicles was compared with that in methanol, demonstrating that several amide sites were protected from exchange by the lipid membrane. We also show that hydrophobic peptides can be detected by ESI-MS in the presence of a large molar excess of the detergent Triton X-100. The rate of HDX of M2-TM in Triton X-100 micelles was faster than that in DMPC vesicles but slower than when the peptide had been denatured in methanol. These results indicate that the accessibility of backbone amide sites to the solvent can be profoundly affected by membrane protein structure and dynamics, as well as the properties of model bilayer systems.
Hansen
Raino K
RK
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Broadhurst
R William
RW
Skelton
Paul C
PC
Arkin
Isaiah T
IT
D002498:Centrifugation; D003902:Detergents; D003903:Deuterium / Q000737:chemistry; D005612:Freeze Drying; D006859:Hydrogen / Q000737:chemistry; D008027:Light; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008567:Membranes, Artificial; D000432:Methanol / Q000737:chemistry; D008823:Micelles; D008854:Microscopy, Electron; D010455:Peptides / Q000737:chemistry*; D012542:Scattering, Radiation; D021241:Spectrometry, Mass, Electrospray Ionization
D003902:Detergents; D008055:Lipids; D008565:Membrane Proteins; D008567:Membranes, Artificial; D008823:Micelles; D010455:Peptides; D006859:Hydrogen; D003903:Deuterium; D000432:Methanol