Noncovalent mass spectrometry (MS) is emerging as an invaluable technique to probe the structure, interactions, and dynamics of membrane proteins (MPs). However, maintaining native-like MP conformations in the gas phase using detergent solubilized proteins is often challenging and may limit structural analysis. Amphipols, such as the well characterized A8-35, are alternative reagents able to maintain the solubility of MPs in detergent-free solution. In this work, the ability of A8-35 to retain the structural integrity of MPs for interrogation by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is compared systematically with the commonly used detergent dodecylmaltoside. MPs from the two major structural classes were selected for analysis, including two β-barrel outer MPs, PagP and OmpT (20.2 and 33.5 kDa, respectively), and two α-helical proteins, Mhp1 and GalP (54.6 and 51.7 kDa, respectively). Evaluation of the rotationally averaged collision cross sections of the observed ions revealed that the native structures of detergent solubilized MPs were not always retained in the gas phase, with both collapsed and unfolded species being detected. In contrast, ESI-IMS-MS analysis of the amphipol solubilized MPs studied resulted in charge state distributions consistent with less gas phase induced unfolding, and the presence of lowly charged ions which exhibit collision cross sections comparable with those calculated from high resolution structural data. The data demonstrate that A8-35 can be more effective than dodecylmaltoside at maintaining native MP structure and interactions in the gas phase, permitting noncovalent ESI-IMS-MS analysis of MPs from the two major structural classes, while gas phase dissociation from dodecylmaltoside micelles leads to significant gas phase unfolding, especially for the α-helical MPs studied.

Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization-ion mobility spectrometry-mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120% D2O. In (1)H-(1)H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6% of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.

Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles. Using quantitative mass spectrometry analyses, we show that fast photochemical oxidation reveals differences in the extent of solvent accessibility of residues between the A8-35 and DDM solubilized states, providing a rationale for the increased stability of membrane proteins solubilized with amphipol compared with detergent micelles, as a result of additional intermolecular contacts. Graphical Abstractᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1421-1) contains supplementary material, which is available to authorized users.

Graphical abstract