Electrospray ionization mass spectrometry as a tool to analyze hydrogen/deuterium exchange kinetics of transmembrane peptides in lipid bilayers.
A method is described to study the precise positioning of transmembrane peptides in a phospholipid bilayer combining hydrogen/deuterium (H/D) exchange and nanoelectrospray ionization mass spectrometry. The method was tested by using model systems consisting of designed alpha-helical transmembrane peptides [acetylGW(2)(LA)(5)W(2)Aethanolamine (WALP16) and acetyl-(GA)(3)W(2)(LA)(5)W(2)(AG)(3)ethanolamine (WALP16(+10))] incorporated in large unilamellar vesicles of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine. Both peptides consist of an alternating leucine/alanine hydrophobic core sequence flanked by tryptophan residues as interfacial anchor residues. In the case of WALP16(+10), this sequence is extended at both ends by 5-aa glycine/alanine tails extending into the aqueous phase surrounding the bilayer. H/D exchange of labile hydrogens in these peptides was monitored in time after dilution of the vesicles in buffered deuterium oxide. It was found that the peptides can be measured by direct introduction of the proteoliposome suspension into the mass spectrometer. Several distinct H/D exchange rates were observed (corresponding to half-life values varying from </=2 to approximately 2 x 10(4) min). Fast exchange rates were assigned to the water-exposed tails of WALP16(+10). For both WALP16 and WALP16(+10), intermediate exchange rates were assigned to the residues close to the membrane/water interface, and the slow exchange rates to the membrane-embedded hydrophobic core. These assignments were confirmed by results from collision-induced dissociation tandem mass spectrometry experiments, which allowed analysis of exchange of individual peptide amide linkages. This proteoliposome nanoelectrospray ionization mass spectrometry technique is shown to be an extremely sensitive and powerful tool for revealing site-specific information on peptide-membrane interactions.
Demmers
J A
JA
Department of Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Haverkamp
J
J
Heck
A J
AJ
Koeppe
R E
RE
Killian
J A
JA
D000595:Amino Acid Sequence; D003903:Deuterium / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010455:Peptides; D006859:Hydrogen; D003903:Deuterium