The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.
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A Nanodisc-solubilized membrane protein library is formed by extracting a population of membrane proteins into detergent and then incorporating these proteins into a heterogeneous Nanodisc library, which models the membrane proteome 相似文献
Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.
The gas-phase structures of protein ions have been studied by electron transfer dissociation (ETD) and collision-induced dissociation (CID) after electrospraying these proteins from native-like solutions into a quadrupole ion trap mass spectrometer. Because ETD can break covalent bonds while minimally disrupting noncovalent interactions, we have investigated the ability of this dissociation technique together with CID to probe the sites of electrostatic interactions in gas-phase protein ions. By comparing spectra from ETD with spectra from ETD followed by CID, we find that several proteins, including ubiquitin, CRABP I, azurin, and β-2-microglobulin, appear to maintain many of the salt bridge contacts known to exist in solution. To support this conclusion, we also performed calculations to consider all possible salt bridge patterns for each protein, and we find that the native salt bridge pattern explains the experimental ETD data better than nearly all other possible salt bridge patterns. Overall, our data suggest that ETD and ETD/CID of native protein ions can provide some insight into approximate location of salt bridges in the gas phase.
The effects of binding two small-molecule inhibitors to Agrobacterium sp. strain ATCC 21400 (Abg) β-glucosidase on the conformations and stability of gas-phase ions of Abg have been investigated. Biotin-iminosugar conjugate (BIC) binds noncovalently to Abg while 2,4-dinitro-2-deoxy-2-fluoro-β-d-glucopyranoside (2FG-DNP) binds covalently with loss of DNP. In solution, Abg is a dimer. Mass spectra show predominantly dimer ions, provided care is taken to avoid dissociation of dimers in solution and dimer ions in the ion sampling interface. When excess inhibitor, either covalent or noncovalent, is added to solutions of Abg, mass spectra show peaks almost entirely from 2:2 inhibitor-enzyme dimer complexes. Tandem mass spectrometry experiments show similar dissociation channels for the apo-enzyme and 2FG-enzyme dimers. The +21 dimer produces +10 and +11 monomers. The internal energy required to dissociate the +21 2FG-enzyme to its monomers (767?±?30 eV) is about 36 eV higher than that for the apo-enzyme dimer (731?±?6 eV), reflecting the stabilization of the free enzyme dimer by the 2FG inhibitor. The primary dissociation channels for the noncovalent BIC-enzyme dimer are loss of neutral and charged BIC. The internal energy required to induce loss of BIC is 482?±?8 eV, considerably less than that required to dissociate the dimers. For a given charge state, ions of the covalent and noncovalent complexes have about 15 % and 25 % lower cross sections, respectively, compared with the apo-enzyme. Thus, binding the inhibitors causes the gas-phase protein to adopt more compact conformations. Noncovalent binding surprisingly produces the greatest change in protein ion conformation, despite the weaker inhibitor binding.
We studied the optical properties of gas-phase polysaccharides (maltose, maltotetraose, and maltohexaose) ions by action spectroscopy using the coupling between a quadrupole ion trap and a vacuum ultraviolet (VUV) beamline at the SOLEIL synchrotron radiation facility (France) in the 7 to 18 eV range. The spectra provide unique benchmarks for evaluation of theoretical data on electronic transitions of model carbohydrates in the VUV range. The effects of the nature of the charge held by polysaccharide ions on the relaxation processes were also explored. Finally the effect of isomerization of polysaccharides (with melezitose and raffinose) on their photofragmentation with VUV photons is presented.
Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.
The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions (‘spikes’) on their surface and are unique in having either T?=?3 or T?=?4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the ‘α-determinant’) or in the ‘floor’ regions between them (the ‘β-determinant’). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the β-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.
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Monitoring the binding of the antigen-binding domain (Fab) of mAb 3120 to hepatitis B capsids by native MS and GEMMA 相似文献
Tandem mass spectrometry is a well-established analytical tool for rapid and reliable characterization of oligonucleotides (ONs) and their gas-phase dissociation channels. The fragmentation mechanisms of native and modified nucleic acids upon different mass spectrometric activation techniques have been studied extensively, resulting in a comprehensive catalogue of backbone fragments. In this study, the fragmentation behavior of highly charged oligodeoxynucleotides (ODNs) comprising up to 15 nucleobases was investigated. It was found that ODNs exhibiting a charge level (ratio of the actual to the total possible charge) of 100% follow significantly altered dissociation pathways compared with low or medium charge levels if a terminal pyrimidine base (3' or 5') is present. The corresponding product ion spectra gave evidence for the extensive loss of a cyanate anion (NCO–), which frequently coincided with the abstraction of water from the 3'- and 5'-end in the presence of a 3'- and 5'-terminal pyrimidine nucleobase, respectively. Subsequent fragmentation of the M-NCO– ion by MS3 revealed a so far unreported consecutive excision of a metaphosphate (PO3–)-ion for the investigated sequences. Introduction of a phosphorothioate group allowed pinpointing of PO3– loss to the ultimate phosphate group. Several dissociation mechanisms for the release of NCO– and a metaphosphate ion were proposed and the validity of each mechanism was evaluated by the analysis of backbone- or sugar-modified ONs.
Coarse-grained simulations with charge hopping were performed for a positively charged tetrameric transthyretin (TTR) protein complex with a total charge of +20. Charges were allowed to move among basic amino acid sites as well as N-termini. Charge distributions and radii of gyration were calculated for complexes simulated at two temperatures, 300 and 600 K, under different scenarios. One scenario treated the complex in its normal state allowing charge to move to any basic site. Another scenario blocked protonation of all the N-termini except one. A final scenario used the complex in its normal state but added a basic-site containing tether (charge tag) near the N-terminus of one chain. The differences in monomer unfolding and charging were monitored in all three scenarios and compared. The simulation results show the importance of the N-terminus in leading the unfolding of the monomer units; a process that follows a zipper-like mechanism. Overall, experimentally modifying the complex by adding a tether or blocking the protonation of N-termini may give the potential for controlling the unraveling and subsequent dissociation of protein complexes.
A surface-assisted laser desorption/ionization (SALDI) source is coupled to the Orbitrap mass analyzer; the instrumental approach is tested for the analysis of rhenium (Re) and osmium (Os) complexes with 8-mercaptoquinoline. Silicon (Si) material obtained by laser treatment of monocrystalline Si is used as SALDI substrate. All studied complexes are detected as radical cations, with no protonated molecules. The comparison of SALDI, matrix-assisted laser desorption/ionization (MALDI), and direct laser desorption/ionization (LDI) on metal plates in the same instrumental setup demonstrated that the detection of the studied complexes using SALDI provides the highest sensitivity. The ability to analyze samples rapidly, high purity of spectra, and good analytical parameters make SALDI coupled to the Orbitrap mass analyzer a potentially powerful tool for the detection of Re and Os complexes and related organic, UV-absorbing compounds.
Native mass spectra of large, polydisperse biomolecules with repeated subunits, such as lipoprotein Nanodiscs, can often be challenging to analyze by conventional methods. The presence of tens of closely spaced, overlapping peaks in these mass spectra can make charge state, total mass, or subunit mass determinations difficult to measure by traditional methods. Recently, we introduced a Fourier Transform-based algorithm that can be used to deconvolve highly congested mass spectra for polydisperse ion populations with repeated subunits and facilitate identification of the charge states, subunit mass, charge-state-specific, and total mass distributions present in the ion population. Here, we extend this method by investigating the advantages of using overtone peaks in the Fourier spectrum, particularly for mass spectra with low signal-to-noise and poor resolution. This method is illustrated for lipoprotein Nanodisc mass spectra acquired on three common platforms, including the first reported native mass spectrum of empty “large” Nanodiscs assembled with MSP1E3D1 and over 300 noncovalently associated lipids. It is shown that overtone peaks contain nearly identical stoichiometry and charge state information to fundamental peaks but can be significantly better resolved, resulting in more reliable reconstruction of charge-state-specific mass spectra and peak width characterization. We further demonstrate how these parameters can be used to improve results from Bayesian spectral fitting algorithms, such as UniDec.
An increasing number of fluorinated drugs, pesticides, and fine chemicals are now produced and applied, especially those containing polyfluorinated aromatic moieties. However, at present, the extent of literature covering the special mass spectrometric behaviors of these compounds remains limited. Herein, we report an unexpected but also general gas-phase dissociation mode of polyfluorinated aromatics in mass spectrometry: expulsion of difluorocarbene (50-Da neutral loss). Results from accurate mass measurements, tandem mass spectrometric experiments, and density functional theory (DFT) calculations support an intramolecular F-atom “ring-walk” migration mechanism for gas-phase CF2 loss. Based on an assessment of the electron ionization-mass spectrometry (EI-MS) data of more than 40 polyfluorinated aromatic compounds from the National Institute of Standards and Technology data bank, we generalized on the substitution group effects on the difluorocarbene dissociation process of polyfluorinated aromatic compounds in EI-MS. These studies have enriched our knowledge of the special gas-phase reactivity of polyfluorinated aromatics and will provide valuable information in further analytical research of these compounds by mass spectrometry.
Top-down approaches for the characterization of intact proteins and macromolecular complexes are becoming increasingly popular, since they potentially simplify and speed up the assignment process. Here we demonstrate how, on a commercially available Q-TWIMS-TOF instrument, we performed top-down ETD of the native form of tetrameric alcohol dehydrogenase. We achieved good sequence coverage throughout the first 81 N-terminal amino acids of ADH, with the exception of a loop located on the inside of the protein. This is in agreement with the exposed parts of the natively folded protein according to the crystal structure. Choosing the right precursor charge state and applying supplemental activation were found to be key to obtaining a high ETD fragmentation efficiency. Finally, we briefly discuss opportunities to further increase the performance of ETD based on our results.
The mechanism of reactions occurring in solution can be investigated also in the gas phase by suited mass spectrometric techniques, which allow to highlight fundamental mechanistic features independent of the influence of the medium and to clarifying controversial hypotheses proposed in solution studies. In this work, we report a gas-phase study performed by electrospray triple stage quadrupole mass spectrometry (ESI-TSQ/MS) on the dehydration of d-xylose, leading mainly to the formation of 2-furaldehyde (2-FA). It is generally known in carbohydrate chemistry that the thermal acid catalyzed dehydration of pentoses leads to the formation of 2-FA, but several aspects on the solution-phase mechanism are controversial. Here, gaseous reactant ions corresponding to protonated xylose molecules obtained from ESI of a solution containing d-xylose and ammonium acetate as protonating reagent were allowed to undergo collisionally activated decomposition (CAD) into the triple stage quadrupole analyzer. The product ion mass spectra of protonated xylose are characterized by the presence of ionic intermediates arising from xylose dehydration, which were structurally characterized by their fragmentation patterns. As expected, the xylose triple dehydration leads to the formation of the ion at m/z 97, corresponding to protonated 2-FA. On the basis of mass spectrometric evidences, we demonstrated that in the gas phase, the formation of 2-FA involves protonation at the OH group bound to the C1 atom of the sugar, the first ionic intermediate being characterized by a cyclic structure. Finally, energy resolved product ion mass spectra allowed to obtain information on the energetic features of the d-xylose→2-FA conversion.
The fragmentations of [AA + M]+ complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag?).
Caption for Graphical Abstract
CID products of Li+, Na+, and Ag+ complexes of Phe, Tyr, Trp, and His are reported and mechanisms by which they are formed are proposed. 相似文献
The gas-phase structures of alkali metal cation-cytosine complexes generated by electrospray ionization are probed via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra of five alkali metal cation–cytosine complexes exhibit both similar and distinctive spectral features over the range of ~1000–1900 cm-1. The IRMPD spectra of the Li+(cytosine), Na+(cytosine), and K+(cytosine) complexes are relatively simple but exhibit changes in the shape and shifts in the positions of several bands that correlate with the size of the alkali metal cation. The IRMPD spectra of the Rb+(cytosine) and Cs+(cytosine) complexes are much richer as distinctive new IR bands are observed, and the positions of several bands continue to shift in relation to the size of the metal cation. The measured IRMPD spectra are compared to linear IR spectra of stable low-energy tautomeric conformations calculated at the B3LYP/def2-TZVPPD level of theory to identify the conformations accessed in the experiments. These comparisons suggest that the evolution in the features in the IRMPD action spectra with the size of the metal cation, and the appearance of new bands for the larger metal cations, are the result of the variations in the intensities at which these complexes can be generated and the strength of the alkali metal cation-cytosine binding interaction, not the presence of multiple tautomeric conformations. Only a single tautomeric conformation is accessed for all five alkali metal cation–cytosine complexes, where the alkali metal cation binds to the O2 and N3 atoms of the canonical amino-oxo tautomer of cytosine, M+(C1).
The gas-phase reactions of a series of coordinatively unsaturated [Ni(L)n]y+ complexes, where L is a nitrogen-containing ligand, with chemical warfare agent (CWA) simulants in a miniature rectilinear ion trap mass spectrometer were investigated as part of a new approach to detect CWAs. Results show that upon entering the vacuum system via a poly(dimethylsiloxane) (PDMS) membrane introduction, low concentrations of several CWA simulants, including dipropyl sulfide (simulant for mustard gas), acetonitrile (simulant for the nerve agent tabun), and diethyl phosphite (simulant for nerve agents sarin, soman, tabun, and VX), can react with metal complex ions generated by electrospray ionization (ESI), thereby providing a sensitive means of detecting these compounds. The [Ni(L)n]2+ complexes are found to be particularly reactive with the simulants of mustard gas and tabun, allowing their detection at low parts-per-billion (ppb) levels. These detection limits are well below reported exposure limits for these CWAs, which indicates the applicability of this new approach, and are about two orders of magnitude lower than electron ionization detection limits on the same mass spectrometer. The use of coordinatively unsaturated metal complexes as reagent ions offers the possibility of further tuning the ion-molecule chemistry so that desired compounds can be detected selectively or at even lower concentrations.
Structural characterization of protonated gas-phase ions of cysteine and dopamine by infrared multiple photon dissociation (IRMPD) spectroscopy using a free electron laser in combination with theory based on DFT calculations reveals the presence of two types of protonated dimer ions in the electrospray mass spectra of the metabolites. In addition to the proton-bound dimer of each species, the covalently bound dimer of cysteine (bound by a disulfide linkage) has been identified. The dimer ion of m/z 241 observed in the electrospray mass spectra of cysteine has been identified as protonated cystine by comparison of the experimental IRMPD spectrum to the IR absorption spectra predicted by theory and the IRMPD spectrum of a standard. Formation of the protonated covalently bound disulfide-linked dimer ions (i.e. protonated cystine) from electrospray of cysteine solution is consistent with the redox properties of cysteine. Both the IRMPD spectra and theory indicate that in protonated cystine the covalent disulfide bond is retained and the proton is involved in intramolecular hydrogen bonding between the amine groups of the two cysteine amino acid units. For cysteine, the protonated covalently bound dimer (m/z 241) dominated the mass spectrum relative to the proton-bound dimer (m/z 243), but this was not the case for dopamine, where the protonated monomer and the proton-bound dimer were both observed as major ions. An extended conformation of the ethylammonium side chain of gas-phase protonated dopamine monomer was verified from the correlation between the predicted IR absorption spectra and the experimental IRMPD spectrum. Dopamine has the same extended ethylamine side chain conformation in the proton-bound dopamine dimer identified in the mass spectra of electrosprayed dopamine. The structure of the proton-bound dimer of dopamine is confirmed by calculations and the presence of an IR band due to the shared proton. The presence of the shared proton in the protonated cystine ion can be inferred from the IRMPD spectrum.
UV–vis photodissociation action spectroscopy is becoming increasingly prevalent because of advances in, and commercial availability of, ion trapping technologies and tunable laser sources. This study outlines in detail an instrumental arrangement, combining a commercial ion-trap mass spectrometer and tunable nanosecond pulsed laser source, for performing fully automated photodissociation action spectroscopy on gas-phase ions. The components of the instrumentation are outlined, including the optical and electronic interfacing, in addition to the control software for automating the experiment and performing online analysis of the spectra. To demonstrate the utility of this ensemble, the photodissociation action spectra of 4-chloroanilinium, 4-bromoanilinium, and 4-iodoanilinium cations are presented and discussed. Multiple photoproducts are detected in each case and the photoproduct yields are followed as a function of laser wavelength. It is shown that the wavelength-dependent partitioning of the halide loss, H loss, and NH3 loss channels can be broadly rationalized in terms of the relative carbon-halide bond dissociation energies and processes of energy redistribution. The photodissociation action spectrum of (phenyl)Ag2+ is compared with a literature spectrum as a further benchmark.
