In this study the gas-phase conformer preferences of Gramicidin A (GA), a linear antimicrobial pentadecapeptide, were investigated directly from aqueous solutions of lipid vesicle bilayers using a mixing tee-electrospray ionization (MT-ESI) setup coupled with ion mobility mass spectrometry (IM-MS). The required time for GA sample preparation was decreased by approximately 50% using MT-ESI when compared to previously reported methods which required freeze-drying of samples. Using an MT-ESI approach to analyze samples of GA associated with POPC (16:0, 18:1 PC) and DEPC (22:1 PC) lipid bilayers yielded dimer conformer preferences comparable to results obtained using more lengthy protocols. GA analogues that contain leucine to lysine substitutions were analyzed; these analogues yielded more hydrophilic GA dimers owing to the hydrophilicity of lysine head groups. The conformer preferences of lipid bilayer associated hydrophilic GA analogues can be obtained owing to disassociation of lipids during the fast mixing time MT-ESI process. The data for both GA analogues associated with negatively charged POPC/POPG (16:0, 18:1 PC/PG) lipid bilayers reveal a preference for antiparallel double helix (ADH) formation. The adoption of nascent conformers for both GA analogues was observed using MT-ESI for samples associated with DMPC/DMPG (12:0 PC/PG) bilayers.

ConspectusElectrospray ionization (ESI) combined with ion mobility-mass spectrometry (IM-MS) is adding new dimensions, that is, structure and dynamics, to the field of biological mass spectrometry. There is increasing evidence that gas-phase ions produced by ESI can closely resemble their solution-phase structures, but correlating these structures can be complicated owing to the number of competing effects contributing to structural preferences, including both inter- and intramolecular interactions. Ions encounter unique hydration environments during the transition from solution to the gas phase that will likely affect their structure(s), but many of these structural changes will go undetected because ESI–IM-MS analysis is typically performed on solvent-free ions. Cryogenic ion mobility-mass spectrometry (cryo-IM-MS) takes advantage of the freeze-drying capabilities of ESI and a cryogenically cooled IM drift cell (80 K) to preserve extensively solvated ions of the type [M + xH]x+(H2O)n, where n can vary from zero to several hundred. This affords an experimental approach for tracking the structural evolution of hydrated biomolecules en route to forming solvent-free gas-phase ions. The studies highlighted in this Account illustrate the varying extent to which dehydration can alter ion structure and the overall impact of cryo-IM-MS on structural studies of hydrated biomolecules.Studies of small ions, including protonated water clusters and alkyl diammonium cations, reveal structural transitions associated with the development of the H-bond network of water molecules surrounding the charge carrier(s). For peptide ions, results show that water networks are highly dependent on the charge-carrying species within the cluster. Specifically, hydrated peptide ions containing lysine display specific hydration behavior around the ammonium ion, that is, magic number clusters with enhanced stability, whereas peptides containing arginine do not display specific hydration around the guanidinium ion. Studies on the neuropeptide substance P illustrate the ability of cryo-IM-MS to elucidate information about heterogeneous ion populations. Results show that a kinetically trapped conformer is stabilized by a combination of hydration and specific intramolecular interactions, but upon dehydration, this conformer rearranges to form a thermodynamically favored gas-phase ion conformation. Finally, recent studies on hydration of the protein ubiquitin reveal water-mediated dimerization, thereby illustrating the extension of this approach to studies of large biomolecules. Collectively, these studies illustrate a new dimension to studies of biomolecules, resulting from the ability to monitor snapshots of the structural evolution of ions during the transition from solution to gas phase and provide unparalleled insights into the intricate interplay between competing effects that dictate conformational preferences.

Covalently linked diubiquitin (diUbq) is known to adopt specific interfacial interactions owing to steric hindrance induced by the covalent tether. K48-linked diUbq preferentially forms hydrophobic interfacial interactions between the two I44 faces under physiological conditions, whereas K63-linked diUbq preferentially forms electrostatic interfacial interactions. Here, we show using collision-induced unfolding ion mobility-mass spectrometry that the recently reported noncovalent dimer of ubiquitin exhibits structural preferences and interfacial interactions that are most similar to that of K48-linked diUbq.

Electrospray ionization (ESI) of ubiquitin from acidified (0.1%) aqueous solution produces abundant ubiquitin–chloride adduct ions, [M + nH + xCl](n – x)+, that upon mild heating react via elimination of neutral HCl. Ion mobility collision cross section (CCS) measurements show that ubiquitin ions retaining chloride adducts exhibit CCS values similar to those of the “native-state” of the protein. Coupled with results from recent molecular dynamics (MD) simulations for the evolution of a salt-containing electrospray droplet, this study provides a more complete picture for how the presence of salts affects the evolution of protein conformers in the final stages of dehydration of the ESI process and within the instrument.

When the all-cis polyproline-I helix (PPI, favored in 1-propanol) of polyproline-13 is introduced into water, it folds into the all-trans polyproline-II (PPII) helix through at least six intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702–12711 (2014)]. Here, we show that the solvent-free intermediates refold into the all-cis PPI helix with high (>90%) efficiency. Moreover, in the absence of solvent, each intermediate appears to utilize the same small set of pathways observed for the solution-phase PPII → PPI transition upon immersion of PPIIaq in 1-propanol. That folding in solution (under conditions where water is displaced by propanol) and folding in vacuo (where energy required for folding is provided by collisional activation) occur along the same pathway is remarkable. Implicit in this statement is that 1-propanol mimics a “dry” environment, similar to the gas phase. We note that intermediates with structures that are similar to PPIIaq can form PPII under the most gentle activation conditions—indicating that some transitions observed in water (i.e., “wet” folding, are accessible (albeit inefficient) in vacuo. Lastly, these “dry” folding experiments show that PPI (all cis) is favored under “dry” conditions, which underscores the role of water as the major factor promoting preference for trans proline.

Hydration of the ammonium ion plays a key role in determining the biomolecular structure as well as local structure of water in aqueous environments. Experimental data obtained by cryogenic ion mobility-mass spectrometry (cryo-IM-MS) show that dehydration of alkyl diammonium cations induces a distinct unfolding transition at a critical number of water molecules, n = 21 to 23, n = 24 to 26, and n = 27 to 29, for 1,7-diaminoheptane, 1,8-diaminooctane, and 1,10-diaminodecane, respectively. Results are also presented that reveal compelling evidence for unique structural transitions of hydrated ammonium ions associated with the development of the hydrogen-bond network around individual charged groups. The ability to track the evolution of structure upon stepwise dehydration provides direct insight into the intricate interplay between solvent–molecule interactions that are responsible for defining conformations. Such insights are potentially valuable in understanding how ammonium ion solvation influences conformation(s) of larger biomolecules.

A novel sample preparation method to probe the solution phase structure of dimerized Gramicidin A (GA) inserted into lipid vesicle bilayers is described. This method, termed vesicle capture-freeze-drying (VCFD), when coupled with electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS), successfully demonstrates the first evidence for the preservation of membrane-bound structure in the analysis of solution phase conformers retained into the gas phase. The extremely hydrophobic character of GA ensures that only membrane-bound conformations are captured and subsequently monitored when samples are prepared using VCFD, removing a barrier that has prevented previous attempts at direct analysis using mass spectrometry. Solution-phase physicochemical interactions of GA influenced by lipid acyl chain length and extent of acyl chain unsaturation can now be probed by monitoring the conformer preferences using IM-MS. Increasing the acyl chain length from 12 to 22 carbons yields [2GA + 2Na]2+ IM-MS profiles with reduced conformer microheterogeneity. POPC (16:0, 18:1 PC), a lipid possessing a single acyl chain unsaturation point, yields the highest abundance of the single stranded head to head (SSHH) conformer. Conformer preferences adopted in the lipid bilayer are maintained as GA dimers travel from the solution phase to fully desolvated gas-phase ions demonstrating that distributions observed using ESI-IM-MS unambiguously reflect the ensemble of conformers observed in the solution phase. VCFD-ESI-IM-MS yields novel biophysical insight into the influence of lipid bilayer membranes on conformer preferences and conformer heterogeneity of an important channel-forming membrane peptide.

The effects of charge states, charge sites and side chain interactions on conformational preferences of gas-phase peptide ions are examined by ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulations. Collision cross sections (CCS) of [M + 2H]2+ and [M + 3H]3+ ions for a series of model peptides, viz. Ac-(AAKAA)nY-NH2 (AKn, n = 3–5) and Ac-Y(AEAAKA)nF-NH2 (AEKn, n = 2–5) are measured by using IM-MS and compared with calculated CCS for candidate ions generated by MD simulations. The results show that charge states, charge sites and intramolecular charge solvation are important determinants of conformer preference for AKn and AEKn ions. For AKn ions, there is a strong preference for helical conformations near the N-terminus and charge-solvated conformations near the C-terminus. For [AEKn + 2H]2+ ions, conformer preferences appear to be driven by charge solvation, whereas [AEKn + 3H]3+ ions favor more extended coil-type conformations.

Phosphonates are a large class of organophosphorus compounds with a characteristic carbon–phosphorus bond. The genes responsible for phosphonate utilization in Gram-negative bacteria are arranged in an operon of 14 genes. The carbon–phosphorus lyase complex, encoded by the genes phnGHIJKLM, catalyzes the cleavage of the stable carbon–phosphorus bond of organophosphonates to the corresponding hydrocarbon and inorganic phosphate. Recently, complexes of this enzyme containing five subunits (PhnG-H-I-J-K), four subunits (PhnG-H-I-J), and two subunits (PhnG-I) were purified after expression in Escherichia coli ( Proc. Natl. Acad. Sci., U. S. A. 2011, 108, 11393). Here we demonstrated using mass spectrometry, ultracentrifugation, and chemical cross-linking experiments that these complexes are formed from a PhnG2I2 core that is further elaborated by the addition of two copies each of PhnH and PhnJ to generate PhnG2H2I2J2. This complex adds an additional subunit of PhnK to form PhnG2H2I2J2K. Chemical cross-linking of the five-component complex demonstrated that PhnJ physically interacts with both PhnG and PhnI. We were unable to demonstrate the interaction of PhnH or PhnK with any other subunits by chemical cross-linking. Hydrogen–deuterium exchange was utilized to probe for alterations in the dynamic properties of individual subunits within the various complexes. Significant regions of PhnG become less accessible to hydrogen/deuterium exchange from solvent within the PhnG2I2 complex compared with PhnG alone. Specific regions of PhnI exhibited significant differences in the H/D exchange rates in PhnG2I2 and PhnG2H2I2J2K.

The reaction of cadmium-binding human metallothionein-2A (Cd7MT) and N-ethylmaleimide (NEM) is investigated by electrospray ionization-ion mobility-mass spectrometry (ESI IM-MS). MS provides a direct measure of the distribution of the kinetic intermediates as the reaction proceeds and provides new insights into the relative kinetic stability of the individual metal–thiolate bonds in Cd7MT. The rate constants for the various metal-retaining intermediates (Cdi, intermediate with i Cd2+ ions attached) differ by >3 orders of magnitude: Cd4 ≪ Cd3 < Cd2 < Cd1 ∼ Cd6 < Cd7 < Cd5. The reaction is viewed as a two-component cooperative process, rapid loss of three Cd2+ ions followed by slow loss of the remaining four Cd2+ ions, and Cd4NEM10MT was observed as the least reactive intermediate during the entire displacement process. “MS-CID-IM-MS”, a top-down approach that provides two-dimensional dispersion (size to charge by IM; mass to charge by MS) of the CID fragment ions, was used for direct analysis of the kinetic intermediate [Cd4NEM10MT]5+ ion. The results provide direct evidence that the four Cd2+ ions located in the α-domain are retained, indicative of the greater kinetic stability for the α-domain. Further, the mapping of the alkylation sites in the [Cd4NEM10MT]5+ ion reveals that not only the nine cysteines in the β-domain but Cys33 in the α-domain is selectively labeled. The kinetic lability of the Cd–Cys33 bond is unexpected. The structural and functional implications of these findings are discussed.

The dynamics, structures, and functions of most biological molecules are strongly influenced by the nature of the peptide’s or protein’s interaction with water. Here, cryogenic ion mobility-mass spectrometry studies of ubiquitin have directly captured a water-mediated protein–protein binding event involving hydrated, noncovalently bound dimer ions in solution, and this interaction has potential relevance to one of the most important protein–protein interactions in nature. As solvent is removed, dimer ions, viz. [2 M + 14H]14+, can be stabilized by only a few attached water molecules prior to dissociation into individual monomeric ions. The hydrophobic patch of ubiquitin formed by the side chains of Leu-8, Ile-44, and Val-70 meet all the necessary conditions for a protein–protein binding “hot spot,” including the requirement for occlusion of water to nearby hydrophilic sites, and it is suggested that this interaction is responsible for formation of the hydrated noncovalent ubiquitin dimer.

Substance P (RPKPQQFFGLM-NH2) [M + 3H]3+ ions have been shown to occupy two distinct conformer states, a compact population of conformers that is formed by evaporation of hydrated ions, and an elongated population of conformers that is formed by collisional heating of the compact conformer. Molecular dynamics (MD) simulations and amino acid mutations revealed that the compact conformer is stabilized by intramolecular interactions between the localized charge-carrying sites, specifically the N-terminus, R1, and K3, with the side chains of glutamine and phenylalanine residues present in the peptide. Here, we employ amino acid mutations and cryogenic ion mobility-mass spectrometry (cryo-IM-MS) in an effort to understand how eliminating specific intramolecular interactions alters ion hydration, as well as the dehydration dynamics of substance P during the final stages of the electrospray process. The results clearly illustrate a direct link between the stabilizing effects of intramolecular self-solvation and the formation of substance P [M + 3H]3+ ions. Most notably, removal of these stabilizing interactions leads to a reduction in the abundances of [M + 3H]3+ ions induced by charge reduction reactions, i.e., loss of H+(H2O)n ions to form [M + 2H]2+ ions during the final stages of the electrospray process.

Here, we critically evaluate the effects of changes in the ion internal energy (Eint) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the [M + 6H]6+ ion of ubiquitin (ubq6+), the [M + 5H]5+ ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the [CdiMT]5+ ion. The ion-neutral CCS for ions formed by “native-state” ESI show a strong dependence on Eint. Collisional activation is used to increase Eint prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of experimental CCSs with those generated by molecular dynamics (MD) simulation for solution-phase ions and solvent-free ions as a function of temperature provide new insights about conformational preferences and retention of solution conformations. The Eint-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the Eint is increased, these structurally labile conformers unfold to an elongated conformation.

Electron capture dissociation mass spectrometry offers several advantages for the analysis of peptides, most notably that backbone c and z fragments typically retain labile modifications such as phosphorylation. We have shown previously that, in some cases, the presence of phosphorylation has a deleterious effect on peptide sequence coverage, and hypothesized that intramolecular interactions involving the phosphate group were preventing separation of backbone fragments. In the present work, we seek to rationalize the observed ECD behavior through a combination of ECD of model peptides, traveling wave ion mobility mass spectrometry and molecular dynamics simulations. The results suggest that for doubly protonated ions of phosphopeptide APLpSFRGSLPKSYVK a salt-bridge structure is favored, whereas for the doubly-protonated ions of APLSFRGSLPKpSYVK ionic hydrogen bonds predominate.

A porous silver-nanoparticle (AgNP)-embedded thin film biosensor was produced by the sol–gel method. The thin films were used as matrix-free laser desorption ionization mass spectrometry (LDI-MS) biosensors applicable to several chemical classes. In these experiments, UV laser irradiation (337 nm) of the AgNP facilitates desorption and ionization of a number of peptides, triglycerides, and phospholipids. Preferential ionization of sterols from vesicles composed of olefinic phosphosphatidylcholines is also demonstrated, offering the possibility for a simplified approach for sterol analysis from complex mixtures. The composition of the nanoparticles was confirmed by X-ray photoelectron spectroscopy (XPS) and UV–vis spectroscopy. XPS data revealed a binding energy of 368.2 eV, consistent with the previous assignment of the binding energy for the Ag 3d5/2 peak from Ag0 at 368.1 ± 0.1 eV. The surface morphology of the thin films was studied by field-emission scanning electron microscopy (FE-SEM) and revealed the presence of nanoparticles and the porous nature of the biosensor.

The methodology for obtaining accurate ion-neutral collision cross section (Ω) values for peptides and proteins using periodic focusing ion mobility spectrometry (PF IMS) is presented. A mobility dampening factor (represented by the term α) is introduced to account for the relative increase in ion-neutral collisions in PF IMS compared to uniform field ion mobility spectrometry (UF IMS) for equivalent operating conditions. The results show that α may be easily quantified both theoretically and empirically for a specific PF IMS design operating at a given pressure based upon the charge state of the analyte. By simply incorporating an α term into traditional UF IMS expressions, accurate Ω values were obtained with excellent agreement (≤4% difference) compared to UF IMS measurements found in the current literature.

The fabrication of a label-free mass spectrometry and optical detection-based biosensor platform for the detection of low-abundance lipophilic analytes in complex mixtures is described. The biosensor consists of a lipid layer partially tethered to the surface of a gold nanorod. The effectiveness of the biosensor is demonstrated for the label-free detection of a lipophilic drug in aqueous solution and a lipopeptide in serum.

The purpose of this work is to expand on the theory presented by Silveira et al. [Silveira et al., International Journal of Mass Spectrometry 296 (2010) 36–42], to include a detailed discussion of discrete ion transport properties in the periodic-focusing DC ion guide (PDC IG) that result in radial ion focusing and ion mobility. We previously noted that although the PDC IG utilizes only electrostatic fields, ions are subjected to an effective RF as they traverse the device in the axial (z) direction. Here, the radial electric field (Er) oscillations generating the effective RF are investigated in detail. Equations of motion are derived to explain ion movement in the radial (r) direction. The results suggest that a collisionally dampened effective potential (V*) model can explain the observed radial ion confinement. Furthermore, a mathematical explanation regarding the effects of the non-uniform axial electric field and periodic collisional cooling phenomena generated in the PDC IG is presented in the context of ion mobility spectrometry (IMS). Included is a detailed discussion of the ion mobility coefficient (K), ion mobility resolution (R), and subsequent determination of the ion-neutral collision cross section (Ω) using the PDC IG. The results indicate that the PDC IG affords straightforward and accurate determination of K and Ω via incorporation of a mobility damping coefficient (α) which is easily derived based upon the operating conditions and the electrode geometry.Graphical abstract.Research highlights► Discrete ion transport modes in a PDC IG (axial drift, radial ripple, and central drift motion) are deconvoluted and discussed. ► Equations of motion are derived to mathematically explain ion motion in the axial and radial directions. ► The results support the radial focusing model based on a collisionally dampened effective potential. ► Derivation of ion-neutral collision cross sections using first-order IMS principles is discussed.

The resolution of ion mobility spectrometry (IMS) is of paramount importance for both post-ionization separations and structural characterization of ions that have similar ion-neutral collision cross sections; however, the instrumental features that lead to increased resolution also decrease ion transmission through the drift cell. The periodic-focusing DC ion guide (PDC IG) drift cell provides increased ion transmission with minimal loss in resolution.In earlier work we showed that the electrode geometry (inner diameter, thickness, and spacing) strongly affects ion focusing and ion transmission. Here, we critically evaluate the effect of the electrode geometry of a PDC IG drift cell on both ion transmission and resolution. In this study we examine two drift cells that differ in length (63 and 125 cm) and electrode configuration. We also examine the effects of applied voltage and pressure in an attempt to maximize both resolution and ion transmission. Experimental data obtained with fullerene and model peptide ions are compared with calculated ion trajectories using SIMION 8.0 simulations.Graphical abstractAn investigation of increased length periodic-focusing DC ion guide (PDC IG) drift cells is presented. The PDC IG drift cell provides increased ion transmission with minimal loss in resolution compared with conventional uniform electric field designs.Research highlights▶ Electrode geometry effects on ion transmission and mobility resolution for a periodic-focusing DC ion guide (PDC IG) drift cell. ▶ Increased ion transmission with a PDC IG drift cell with minimal degradation in mobility resolution. ▶ Ion mobility resolution increase on increased length PDC IG drift cell agrees with diffusion limited resolving power equation without a decrease in ion transmission.

A hybrid ion mobility-mass spectrometer (IM-MS) incorporating a variable-temperature (80–400 K) drift tube is presented. The instrument utilizes an electron ionization (EI) source for fundamental small molecule studies. Ions are transferred to the IM-MS analyzer stages through a quadrupole, which can operate in either broad transmission or mass-selective mode. Ion beam modulation for the ion mobility experiment is accomplished by an electronic shutter gate. The variable-temperature ion mobility spectrometer consists of a 30.2 cm uniform field drift tube enclosed within a thermal envelope. Subambient temperatures down to 80 K are achievable through cryogenic cooling with liquid nitrogen, while elevated temperatures can be accessed through resistive heating of the envelope. Mobility separated ions are mass analyzed by an orthogonal time-of-flight (TOF) mass spectrometer. This report describes the technological considerations for operating the instrument at variable temperature, and preliminary results are presented for IM-MS analysis of several small mass ions. Specifically, mobility separations of benzene fragment ions generated by EI are used to illustrate significantly improved (greater than 50%) ion mobility resolution at low temperatures resulting from decreased diffusional broadening. Preliminary results on the separation of long-lived electronic states of Ti+ formed by EI of TiCl4 and hydration reactions of Ti+ with residual water are presented.

In this work, we provide a comprehensive understanding of the radial ion focusing mechanism in the periodic-focusing DC ion guide (PDC IG). The PDC IG was developed in our laboratory to improve the sensitivity and throughput of ion mobility spectrometry (IMS) with respect to conventional uniform field IMS. Radial ion focusing, which is responsible for the sensitivity improvement, is attributed to the presence of effective potentials created by the fringing electric fields of thick ring electrodes and collisional cooling of the ions with the neutral buffer gas. The ion focusing mechanism is affirmed by investigating the variations in the effective ion temperature (Teff) which are dependent upon axial position in the device. The concepts derived herein outline guidelines for the design of high performance PDC IG ion mobility instruments and other ion optical devices such as periodic-focusing DC ion funnels.Graphical abstractRadial ion confinement during ion mobility separation in the PDC IG is attributed to the presence of effective potentials at the edges of thick periodic-focusing electrodes.Research highlights▶ The PDC IG is an ion mobility spectrometer that yields high ion transmission via suppression of radial diffusion. ▶ Ions are confined in the radial dimension by effective potentials which are present at the electrode edges. ▶ The axial electric field, assisted by collisional cooling, dampens ion kinetic energy near the back edge of electrodes to allow effective potentials to direct ion motion.

A “strategy” for analyte capture/ionization based on chemical derivatization of gold nanorods and infrared laser desorption ionization (IR-LDI) is described. This is the first example of laser desorption/ionization of biomolecules using gold nanorods irradiated with an IR laser. LDI is performed at wavelengths (1064 nm) that overlap with the longitudinal surface plasmon resonance (LSPR) mode of gold nanorods. The absorbed energy from the laser facilitates desorption and ionization of the analyte. The wavelength of the LSPR band can be tuned by controlling the aspect ratio (length-to-diameter) of the nanorod. For example, the SPR band for Au nanorods having an aspect ratio of 5:1 is centered at ∼840 nm, and this band overlaps with the 1064 nm output of a Nd:YAG laser. We show that a variety of biomolecules can be efficiently desorbed and ionized by 1064 nm irradiation of nanorods. We also show that analyte capture can be controlled by surface chemistry of the nanorods. The results of these studies are important for designing nanomaterial-based capture assays for mass spectrometry and interfacing nanomaterials with imaging/spatial profiling mass spectrometry experiments.

The conformational preferences adopted by gramicidin A (GA) dimers inserted into phospholipid bilayers are reported as a function of the bilayer cholesterol content, temperature, and incubation time. Through use of vesicle capture-freeze drying methodology, GA dimers were captured in lipid bilayers and the conformational preferences of the complex were analyzed using ion mobility-mass spectrometry. Perturbations that affect the physicochemical interactions in the lipid bilayer such as cholesterol incorporation, temperature, and incubation time directly alter the conformer preferences of the complex. Regardless of bilayer cholesterol concentration, the antiparallel double helix (ADH) conformation was observed to be most abundant for GA dimers in bilayers composed of lipids with 12 to 22 carbon acyl chains. Incorporation of cholesterol into lipid bilayers yields increased bilayer thickness and rigidity, and an increased abundance of parallel double helix (PDH) and single-stranded head-to-head (SSHH) dimers were observed. Bilayers prepared using 1,2-dilauroyl-sn-glycero-3-phosphocholine, a lipid with 12 carbon acyl chains, yielded a nascent conformer that decreased in abundance as a function of bilayer cholesterol content. High resolution ion mobility-mass spectrometry data revealed two peaks in the ADH region suggesting that ADH populations are composed of two distinct conformers. The conformer preferences of GA dimers from 1,2-distearoyl-sn-glycero-3-phosphocholine bilayers were significantly different for samples incubated at 4°C vs. 60°C; increased cholesterol content yielded more PDH and SSHH at 60°C. The addition of cholesterol as well as incubating samples of 1,2-distearoyl-sn-glycero-3-phosphocholine at 60°C for 24–72 h yielded an increase in PDH and SSHH abundance.