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 pep tides in a phospholipid bilayer combining hydrogen/deuterium (H/D) exchange and nanoelectrospray ionization mass spectrometry. The method was tested b y using model systems consisting of designed alpha-helical transmembrane pe ptides [acetylGW(2)(LA)(5)W(2)Aethanolamine (WALP16) and acetyl(CA)(3)W-2(L A)(5)W-2(AG)(3)ethanolamine (WALP16(+10))] incorporated in large unilamella r Vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Bath peptides co nsist of an alternating leucine/ alanine hydrophobic core sequence flanked by tryptophan residues as interfacial anchor residues, In the case of WALP1 6(+10), this sequence is extended at both ends by 5-aa glycine/alanine tail s 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 c an 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 less than or equal to 2 to a pproximate to 2 x 10(4) min). Fast exchange rates were assigned to the wate r-exposed tails of WALP16(+10), For both WALP16 and WALP16(+10), intermedia te exchange rates were assigned to the residues close to the membrane/water interface, and the slow exchange rates to the membrane-embedded hydrophobi c core. These assignments were confirmed by results from collision-induced dissociation tandem mass spectrometry experiments, which allowed analysis o f exchange of individual peptide amide linkages. This proteoliposome nanoel ectrospray ionization mass spectrometry technique is shown to be an extreme ly sensitive and powerful tool for revealing site-specific information on p eptide-membrane interactions.