Formation and dissociation processes of gas-phase detergent micelles

Growing interest in micelles to protect membrane complexes during the transition from solution to gas phase prompts a better understanding of their properties. We have used ion mobility mass spectrometry to separate and assign detergent clusters formed from the n-trimethylammonium bromide series of detergents. We show that cluster size is independent of detergent concentration in solution, increases with charge state, but surprisingly decreases with alkyl chain length. This relationship contradicts the thermodynamics of micelle formation in solution. However, the liquid drop model, which considers both the surface energy and charge, correlates extremely well with the experimental cluster size. To explore further the properties of gas-phase micelles, we have performed collision-induced dissociation on them during tandem mass spectrometry. We observed both sequential asymmetric charge separation and neutral evaporation from the precursor ion cluster. Interestingly, however, we also found markedly different dissociation pathways for the longer alkyl chain detergents, with significantly fewer intermediate ions formed than for those with a shorter alkyl chain. These experiments provide an essential foundation for understanding the process of the gas-phase analysis of membrane protein complexes. Moreover they imply valuable mechanistic details of the protection afforded to protein complexes by detergent clusters during gas-phase activation processes.     

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.     

A Mass‐Spectrometry‐Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non‐specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid‐binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent‐resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid‐II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non‐annular lipids based on their exchange rates in solution.     

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.     

A new azobenzene-based design strategy for detergents in membrane protein research

Mass spectrometry enables the in-depth structural elucidation of membrane protein complexes, which is of great interest in structural biology and drug discovery. Recent breakthroughs in this field revealed the need for design rules that allow fine-tuning the properties of detergents in solution and gas phase. Desirable features include protein charge reduction, because it helps to preserve native features of protein complexes during transfer from solution into the vacuum of a mass spectrometer. Addressing this challenge, we here present the first systematic gas-phase study of azobenzene detergents. The utility of gas-phase techniques for monitoring light-driven changes of isomer ratios and molecular properties are investigated in detail. This leads to the first azobenzene detergent that enables the native mass spectrometry analysis of membrane proteins and whose charge-reducing properties can be tuned by irradiation with light. More broadly, the presented work outlines new avenues for the high-throughput characterization of supramolecular systems and opens a new design strategy for detergents in membrane protein research.

Here, L. H. Urner and co-workers identify a new detergent design strategy for the non-denaturing structural analysis of membrane proteins by studying the gas-phase properties of azobenzene-based oligoglycerol detergents.     

Nonacid cleavable detergents applied to MALDI mass spectrometry profiling of whole cells

Although cleavable detergents were first synthesized a number of years ago, they have only recently been successfully applied to problems involving biological molecules. Recent reports have demonstrated that these compounds are useful for applications involving both 2D PAGE and mass spectrometry. However, most cleavable surfactants have utilized acid-labile functional groups to affect cleavage. In applications where extreme pH is required, acid cleavable detergents have limited usefulness. We report the synthesis of fluoride cleavable silane compounds and photolabile cinnamate esters as cleavable detergents having alternative cleavage chemistries than previously reported cleavable detergents. These compounds were applied to whole cell analysis using MALDI mass spectrometry, and it was demonstrated that their use results in an increase in the number of proteins analyzed by increasing protein solubility.     

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed.     

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.     

Separation of peptides from detergents using ion mobility spectrometry

Mass spectrometry (MS) has dramatically evolved in the last two decades and has been the driving force of the spectacular expansion of proteomics during this period. However, the very poor compatibility of MS with detergents is still a technical obstacle in some studies, in particular on membrane proteins. Indeed, the high hydrophobicity of membrane proteins necessitates the use of detergents for their extraction and solubilization. Here, we address the analytical potential of high-field asymmetric waveform ion mobility spectrometry (FAIMS) for separating peptides from detergents. The study was focused on peptides from the human integral membrane protein CD9. A tryptic peptide was mixed with the non-ionic detergents Triton X-100 or beta-D-dodecyl maltoside (DDM) as well as with the ionic detergents sodium dodecyl sulfate (SDS) or sodium deoxycholate (SDC). Although electrospray ionization (ESI) alone led to a total suppression of the peptide ion signal on mass spectra with only detection of the detergents, use of FAIMS allowed separation and clear identification of the peptide with any of the detergents studied. The detection and identification of the target compound in the presence of an excess of detergents are then feasible. FAIMS should prove especially useful in the structural and proteomic analysis of membrane proteins.     

Correlating Glycoforms of DC‐SIGN with Stability Using a Combination of Enzymatic Digestion and Ion Mobility Mass Spectrometry

The immune scavenger protein DC‐SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC‐SIGN have not been reported. Herein, we report the development and application of a mass‐spectrometry‐based approach to both uncover and characterise the effects of O‐glycans on the stability of DC‐SIGN. We first quantify the Core 1 and 2 O‐glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O‐glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas‐phase stability of the DC‐SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC‐SIGN is better correlated with the number of glycosylation sites.     

Modern biomolecular mass spectrometry and its role in studying virus structure, dynamics, and assembly

Over a century since its development, the analytical technique of mass spectrometry is blooming more than ever, and applied in nearly all aspects of the natural and life sciences. In the last two decades mass spectrometry has also become amenable to the analysis of proteins and even intact protein complexes, and thus begun to make a significant impact in the field of structural biology. In this Review, we describe the emerging role of mass spectrometry, with its different technical facets, in structural biology, focusing especially on structural virology. We describe how mass spectrometry has evolved into a tool that can provide unique structural and functional information about viral-protein and protein-complex structure, conformation, assembly, and topology, extending to the direct analysis of intact virus capsids of several million Dalton in mass. Mass spectrometry is now used to address important questions in virology ranging from how viruses assemble to how they interact with their host.     

Correlating Glycoforms of DC-SIGN with Stability Using a Combination of Enzymatic Digestion and Ion Mobility Mass Spectrometry

The immune scavenger protein DC-SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC-SIGN have not been reported. Herein, we report the development and application of a mass-spectrometry-based approach to both uncover and characterise the effects of O-glycans on the stability of DC-SIGN. We first quantify the Core 1 and 2 O-glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O-glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas-phase stability of the DC-SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC-SIGN is better correlated with the number of glycosylation sites.     

Probing the Lipid Annular Belt by Gas‐Phase Dissociation of Membrane Proteins in Nanodiscs

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.     

Oscillations in the Stability of Consecutive Chemical Bonds Revealed by Ion‐Induced Desorption

While it is a common concept in chemistry that strengthening of one bond results in weakening of the adjacent ones, no results have been published on if and how this effect protrudes further into the molecular backbone. By binding molecules to a surface in the form of a self‐assembled monolayer, the strength of a primary bond can be selectively altered. Herein, we report that by using secondary‐ion mass spectrometry, we are able to detect for the first time positional oscillations in the stability of consecutive bonds along the adsorbed molecule, with the amplitudes diminishing with increasing distance from the molecule–metal interface. To explain these observations, we have performed molecular dynamics simulations and DFT calculations. These show that the oscillation effects in chemical‐bond stability have a very general nature and break the translational symmetry in molecules.     

Dramatic destabilization of transmembrane helix interactions by features of natural membrane environments

Membrane proteins have evolved to fold and function in a lipid bilayer, so it is generally assumed that their stability should be optimized in a natural membrane environment. Yet optimal stability is not always in accord with optimization of function, so evolutionary pressure, occurring in a complex membrane environment, may favor marginal stability. Here, we find that the transmembrane helix dimer, glycophorin A (GpATM), is actually much less stable in the heterogeneous environment of a natural membrane than it is in model membranes and even common detergents. The primary destabilizing factors are electrostatic interactions between charged lipids and charged GpATM side chains, and nonspecific competition from other membrane proteins. These effects overwhelm stabilizing contributions from lateral packing pressure and excluded volume. Our work illustrates how evolution can employ membrane composition to modulate protein stability.     

Accurate mass measurement and tandem mass spectrometry of intact globin chains identify the low proportion variant hemoglobin Lepore-Boston-Washington from the blood of a heterozygote

Within a mixture of proteins, minor polymorphic components are difficult to identify using a conventional proteomic approach. Their identification generally requires multi-dimensional separation steps, before or after proteolytic cleavage, followed by sequence analysis of the proteolytic products. In this study, we investigated the potential of tandem mass spectrometry for protein characterization by identifying the delta-beta hybrid human hemoglobin variant Lepore-Boston-Washington using electrospray ionization tandem mass spectrometry. Hemoglobin Lepore-Boston-Washington occurs mainly in heterozygotes, where it comprises approximately 10% of the total non-alpha-chains, the dominant non-alpha-chain being the normal beta (approximately 90%). Furthermore, Hemoglobin Lepore-Boston-Washington has an average molecular mass (15,865.23 Da) that is only 2 Da lower than that of the normal beta-chain (15,867.24 Da). Consequently, it cannot be resolved from the normal beta-chain by mass spectrometry. Here we show how Hemoglobin Lepore-Boston-Washington was identified directly from the diluted blood of a heterozygote by analyzing the product ions from the Lepore-Boston-Washington and normal beta-chain ions without prior separation of the individual chains. This study shows the potential of the tandem mass spectrometry for identifying a minor component in an unseparated mixture of proteins.     

A Mass-Spectrometry-Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non-specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid-binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent-resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid-II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution.     

Ion mobility mass spectrometry of two tetrameric membrane protein complexes reveals compact structures and differences in stability and packing

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.     

Membrane protein–surfactant complexes

Transmembrane proteins expose to the surrounding membrane a belt of mainly hydrophobic amino acid residues, which makes them insoluble in water. Solubilizing them and handling them in vitro generally relies on the use of dissociating surfactants (detergents). Exposing membrane proteins to detergents, however, adversely affects their stability, which is a major hindrance in their study. After briefly recalling relevant aspects of membrane protein structure, the modus operandi of detergents and the problems they raise, we describe alternative approaches such as insertion into bicelles or lipid cubic phases, or association with non-detergent amphiphiles such as peptitergents, hemifluorinated surfactants and amphipols. These novel supramolecular assemblies offer a fascinating playground for collaborative studies between organic chemists, physical chemists and biologists, and they have spurred imaginative works in each of these fields.     

DNA stability in the gas versus solution phases: A systematic study of thirty-one duplexes with varying length, sequence, and charge level

We report herein a systematic mass spectrometric study of a series of thirty-one non-self-complementary, matched, DNA duplexes ranging in size from 5- to 12-mers. The purpose of this work is threefold: (1) to establish the viability of using mass spectrometry as a tool for examining solution phase stabilities of DNA duplexes; (2) to systematically assess gas-phase stabilities of DNA duplexes; and (3) to compare gas and solution phase stabilities in an effort to understand how media affects DNA stability. These fundamental issues are of importance both on their own, and also for harnessing the potential of mass spectrometry for biological applications. We have found that ion abundances do not always track with solution phase stability; GC content must be taken into account. Two duplexes with the same Tm yet with differing GC content can yield different ion abundances. That is, if two duplexes have the exact same melting temperature, yet one has a higher GC content, the duplex with the higher GC content yields a higher ion abundance. It thus appears that not only is a GC base pair stronger than an AT base pair, but the relative strengths of each differ in the gas phase versus in solution, such that the electrospray process can differentiate between them. We also characterize the gas-phase stabilities of the duplexes, using collision-induced dissociation (CID) as a method to assess stability. We focus on two aspects of this CID experiment. One, we examine what factors appear to control whether the duplexes dissociate into single strands or covalently fragment; we are able to utilize a charge state normalization we coin "charge level" to compare our results with others' and establish generalities regarding dissociation versus fragmentation patterns. Two, we examine those duplexes that primarily dissociate and use CID to assess the gas-phase stabilities. We find that correlation of gas-phase to solution-phase stabilities is more likely to occur when duplexes of varying GC content are examined. Duplexes with the same GC content tend to have stabilities that do not parallel those in solution. We discuss these results in light of the different roles that hydrogen bonding and base stacking play in solution versus the gas phase. Ultimately, we apply what we learn to lend insight into the biological problem of how the carcinogenic, damaged nucleobase O6-methylguanine causes mutations.