Drug Binding Revealed by Tandem Mass Spectrometry of a Protein-Micelle Complex Leopold L. Ilag, ² Iban Ubarretxena-Belandia, ‡ Christopher G. Tate, ‡ and Carol V. Robinson* ,² Department of Chemistry, UniVersity of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK, and MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK Received August 18, 2004; E-mail: cvr24@cam.ac.uk Electrospray mass spectrometry (ES MS) approaches for char- acterizing membrane proteins in general involve extraction into organic solvents to maintain solubility and enable spectra of unfolded protein molecules to be recorded. 1 Consequently, under such conditions, interactions between proteins and lipids are lost. Strategies that have been developed to overcome these denaturing approaches involve various lipid preparations and have led to MS of peripheral and transmembrane proteins bound to lipid mol- ecules 2,3 as well as observation of an intact membrane protein trimer. 4 It has also been reported that clusters of up to 12 sodium dodecylsulfate molecules, derived from an intact micelle, could be preserved using ES MS. 5 Protein-lipid interactions within a protein-micelle complex have not been demonstrated previously. Given that it is possible to obtain ES spectra of megadalton particles such as viruses 6,7 and ribosomes, 8 mass and complexity should not preclude study of protein-micelle complexes. It is established that transit from solution phase to low vacuum devoid of solvent molecules is accompanied by significant increase in electrostatic interactions and concomitant reduction in hydrophobic forces. 9 Consequently, this force that drives micelle formation and contributes to stability in solution is expected to be weakened. Previous ES MS experiments have shown that the hydrophobic nature of membrane proteins and absence of formal charge on nonionic lipid molecules lead to low charge states. 2 The combina- tions of low charge states, as well as the established dynamic nature in solution and potential instability of micelles in the gas phase, represent significant challenges for MS. Here we show that it is possible to transfer into the gas phase a protein-micelle complex of EmrE from E. coli solubilized with the detergent dodecylmaltoside (DDM) while maintaining drug binding within the complex. This 110 amino acid transmembrane protein is involved in multidrug transport. The structure of EmrE in DDM with the cation substrate tetraphenyl phosphonium (TPP + ) has recently been shown by cryoelectron microscopy to involve an asymmetric dimer with four transmembrane helices present in each monomer. 10 One TPP + molecule binds simultaneously to two glutamic acid residues, one from each neighboring EmrE mono- mer. 11 We chose to analyze by ES MS solutions of EmrE in DDM in the presence of TPP + at pH8.0 where the off-rate of substrate is established as extremely slow. 11 This complex has also been extensively characterized by a variety of biophysical methods 12 and yielded the highest resolution structures to date. 10 The MS recorded for EmrE purified in DDM (see Supporting Information) is shown in Figure 1A. Three major distributions are observed labeled peaks 1, 2, and 3. The broadness of these distributions is indicative of a heterogeneous assembly of protein, lipids, water/buffer molecules, and counterions. As such, it is not possible to discern the charge states or consequently the mass of these species. To extract information from this spectrum therefore and deter- mine the composition of the broad peaks we isolated defined m/z ranges using a modified time-of-flight MS with a low-frequency quadrupole capable of isolating packets of ions at high m/z values. 13 One such isolation of ions from peak 2 and subsequent acceleration through the collision cell gives rise to the tandem mass spectrum shown in Figure 1B. Two distinct series of peaks at higher m/z values than the isolated region are observed. Unlike the spectra of proteins or their complexes, neighboring peaks within these series do not differ in charge, rather the difference between each peak is approximately constant, consistent with addition of neutral DDM molecules. These series are assigned therefore to clusters of DDM containing on average 20 ( 2 and 30 ( 1 molecules with one and two charges, respectively. In the low m/z region of the tandem MS, DDM monomers and dimers, as well as fragments, can be discerned. A peak at m/z 2065 ² University of Cambridge. ‡ MRC Laboratory of Molecular Biology. Figure 1. (a) Mass spectrum of EmrE in DDM with TPP + recorded with a needle voltage of 1.7 kV, cone voltage of 90 V, and pressures of 4.5- 8.0, 9.5 × 10 -4 , and 1.7 × 10 -6 mbar in the ion transfer, quadrupole, and ToF analyzers, respectively. (b) MS/MS of ion isolated at 6500 ( 100 m/z units (shaded) and accelerated at 30 V through the collision cell at a pressure of 3.5 × 10 -2 mbar. # represents the peak assigned to TPP + . Inset shows an expansion with ×15 magnification of m/z 2000-2500 showing EmrE 6+ with 1-4 DDM adducts. Published on Web 10/14/2004 14362 9 J. AM. CHEM. SOC. 2004, 126, 14362-14363 10.1021/ja0450307 CCC: $27.50 © 2004 American Chemical Society