Effects of Select Anions from the Hofmeister Series on the Gas-Phase Conformations of Protein Ions Measured with Traveling-Wave Ion Mobility Spectrometry/Mass Spectrometry

The gas-phase conformations of ubiquitin, cytochrome c, lysozyme, and α-lactalbumin ions, formed by electrospray ionization (ESI) from aqueous solutions containing 5 mM ammonium perchlorate, ammonium iodide, ammonium sulfate, ammonium chloride, ammonium thiocyanate, or guanidinium chloride, are examined using traveling-wave ion mobility spectrometry (TWIMS) coupled to time-of-flight (TOF) mass spectrometry (MS). For ubiquitin, cytochrome c, and α-lactalbumin, adduction of multiple acid molecules results in no significant conformational changes to the highest and lowest charge states formed from aqueous solutions, whereas the intermediate charge states become more compact. The transition to more compact conformers for the intermediate charge states occurs with fewer bound H₂SO₄ molecules than HClO₄ or HI molecules, suggesting ion-ion or salt-bridge interactions are stabilizing more compact forms of the gaseous protein. However, the drift time distributions for protein ions of the same net charge with the highest levels of adduction of each acid are comparable, indicating that these protein ions all adopt similarly compact conformations or families of conformers. No significant change in conformation is observed upon the adduction of multiple acid molecules to charge states of lysozyme. These results show that the attachment of HClO₄, HI, or H₂SO₄ to multiply protonated proteins can induce compact conformations in the resulting gas-phase protein ions. In contrast, differing Hofmeister effects are observed for the corresponding anions in solution at higher concentrations.     

An Assessment of Computational Methods for Obtaining Structural Information of Moderately Flexible Biomolecules from Ion Mobility Spectrometry

When utilized in conjunction with modeling, the collision cross section (Ω) from ion mobility spectrometry can be used to deduce the gas phase structures of analyte ions. Gas phase conformations are determined computationally, and their Ω calculated using an approximate method, the results of which are compared with experimental data. Though prior work has focused upon rigid small molecules or large biomolecules, correlation of computational and experimental Ω has not been thoroughly examined for analytes with intermediate conformational flexibility, which constitute a large fraction of the molecules studied in the field. Here, the computational paradigm for calculating Ω has been tested for the tripeptides WGY, YGW, and YWG (Y = tyrosine, W = tryptophan, G = glycine). Experimental data indicate that Ω_exp (YWG) > Ω_exp (WGY) ≈ Ω_exp (YGW). The energy distributions of conformations obtained from tiers of simulated annealing molecular dynamics (SAMD) were analyzed using a wide array of density functionals. These quantum mechanical energy distributions do not agree with the MD data, which leads to structural differences between the SAMD and DFT conformations. The latter structures are obtained by reoptimization of the SAMD geometries, and are the only suite of structures that reproduce the experimental trend in analyte separability. In the absence of fitting Lennard Jones potentials that reproduce experimental results for the Trajectory Method, the Exact Hard Sphere Scattering method produced numerical values that are in best agreement with the experimental cross sections obtained in He drift gas.     

Differentiation of Compact and Extended Conformations of Di-Ubiquitin Conjugates with Lysine-Specific Isopeptide Linkages by Ion Mobility-Mass Spectrometry

Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS–MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS–MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.     

Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: The effect of ligand binding on conformational stability

Ion mobility spectrometry, with subsequent mass spectrometric detection, has been employed to study the stability of compact protein conformations of FK-binding protein, hen egg-white lysozyme, and horse heart myoglobin in the presence and absence of bound ligands. Protein ions, generated by electrospray ionization from ammonium acetate buffer, were activated by collision with argon gas to induce unfolding of their compact structures. The collisional cross sections (Ω) of folded and unfolded conformations were measured in the T-Wave mobility cell of a Waters Synapt HDMS (Waters, Altrincham, UK) employing a calibration against literature values for a range of protein standards. In the absence of activation, collisional cross section measurements were found to be consistent with those predicted for folded protein structures. Under conditions of defined collisional activation energies partially unfolded conformations were produced. The degree of unfolding and dissociation induced by these defined collision energies are related to the stability of noncovalent intra- and intermolecular interactions within protein complexes. These findings highlight the additional conformational stability of protein ions in the gas phase resulting from ligand binding.     

Characterization of acid-induced protein conformational changes and noncovalent complexes in solution by using coldspray ionization mass spectrometry

Coldspray ionization (CSI) mass spectrometry, a variant of electrospray ionization (ESI) operating at low temperature (20 to −80°C), has been used to characterize protein conformation and noncovalent complexes. A comparison of CSI and ESI was presented for the investigation of the equilibrium acid-induced unfolding of cytochrome c, ubiquitin, myoglobin, and cyclophilin A (CypA) over a wide range of pH values in aqueous solutions. CSI and nanoelectrospray ionization (nanoESI) were also compared in their performance to characterize the conformational changes of cytochrome c and myoglobin. Significant differences were observed, with narrower charged-state distribution and a shift to lower charge state in the CSI mass spectra compared with those in ESI and nanoESI mass spectra. The results suggest that CSI is more prone to preserving folded protein conformations in solution than the ESI and nanoESI methods. Moreover, the CSI-MS data are comparable with those obtained by other established biophysical methods, which are generally acknowledged to be the suitable techniques for monitoring protein conformation in solution. Noncovalent complexes of holomyoglobin and the protein-ligand complex between CypA and cyclosporin A (CsA) were also investigated at a neutral pH using the CSI-MS method. The results of this study suggest the ability of CSI-MS in retaining of protein conformation and noncovalent interactions in solution and probing subtle protein conformational changes. Additionally, the CSI-MS method is capable of analyzing quantitatively equilibrium unfolding transitions of proteins. CSI-MS may become one of the promising techniques for investigating protein conformation and noncovalent protein-ligand interactions in solution.     

Elucidating Factors Manipulated the Formation of Multiply Charged Protein Homomultimeric Complexes by Electrospray Ionization

Native non‐covalently bonded protein‐protein and protein‐substrate complexes are of great interest and have been extensively studied by electrospray ionization mass spectrometry (ESI‐MS). Multiply charged protein homomultimeric complexes are shown to form by ESI‐MS. This study addresses factors that can artificially induce the formation of multiply charged protein homomultimeric complexes. Cytochrome c (Cyt c) and ubiquitin, which are monomers in solution, were found to generate (Cyt c)_mⁿ⁺ by electrospray ionization (ESI). The homomultimeric complexes were not limited to dimeric complexes but include also multiply charged trimers, tetramers, and pentamers. The observation of these homomultimeric complexes has never been revealed from a Cyt c solution at the concentration as low as 10 μM. Increasing the concentration of Cyt c enhanced the formation of (Cyt c)_mⁿ⁺ as expected; however, the protein concentration does not affect the relative intensities of monomeric and dimeric complexes. Additionally the enrichment of NH₄OH also promotes the formation of (Cyt c)_mⁿ⁺. Notably, source collision‐induced dissociations (source‐CID) of (Cyt c)_mⁿ⁺ alter the charge state distribution (CSD) and may lead to an incorrect interpretation of Cyt c conformations. Hence, extra care should be taken when using CSD to interpret the conformation of a protein derived from ESI‐MS.     

Identifying conformational changes of the β2 adrenoceptor that enable accurate prediction of ligand/receptor interactions and screening for GPCR modulators

The new β₂ Adrenoceptor (β₂AR) crystal structures provide a high-resolution snapshot of receptor interactions with two particular partial inverse agonists, (−)-carazolol and timolol. However, both experimental and computational studies of GPCR structure are significantly complicated by the existence of multiple conformational states coupled to ligand type and receptor activity. Agonists and antagonists induce or stabilize distinct changes in receptor structure that mediate a range of pharmacological activities. In this work, we (1) established that the existing β₂AR crystallographic conformers can be extended to describe ligand/receptor interactions for additional antagonist types, (2) generated agonist-bound receptor conformations, and (3) validated these models for agonist and antagonist virtual ligand screening (VLS). Using a ligand directed refinement protocol, we derived a single agonist-bound receptor conformation that selectively retrieved a diverse set of full and partial β₂AR agonists in VLS trials. Additionally, the impact of extracellular loop two conformation on VLS was assessed by docking studies with rhodopsin-based β₂AR homology models, and loop-deleted receptor models. A general strategy for constructing and selecting agonist-bound receptor pocket conformations is presented, which may prove broadly useful in creating agonist and antagonist bound models for other GPCRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The study of protein conformation in solution via direct sampling by desorption electrospray ionization mass spectrometry

The direct sampling feature of liquid sample desorption electrospray ionization (DESI) allows the ionization of liquid samples without adding acids/organic solvents (i.e., without sample pretreatment). As a result, it provides a new approach for probing protein conformation in solution. In this study, it has been observed that native protein ions are generated from proteins in water by DESI. Interestingly, the intensities of the resulting protein ions appear to be higher than those generated by ESI of the proteins in water or in ammonium acetate. For protein solutions that already contain acids/organic solvents, DESI can be used to investigate the influences of these denaturants on protein conformations and the obtained results are in good agreement with spectroscopic data. In addition, online monitoring of protein conformational changes by DESI is feasible; for instance, heat-induced unfolding of ubiquitin can be traced with DESI in water without influences of organic solvents/acids. This DESI method provides a new alternative tool for the study of protein conformation in solution.     

Fragmentation methods on the balance: unambiguous top–down mass spectrometric characterization of oxaliplatin–ubiquitin binding sites

The interaction between oxaliplatin and the model protein ubiquitin (Ub) was investigated in a top–down approach by means of high-resolution electrospray ionization mass spectrometry (ESI-MS) using diverse tandem mass spectrometric (MS/MS) techniques, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD). To the best of our knowledge, this is the first time that metallodrug–protein adducts were analyzed for the metal-binding site by ETD-MS/MS, which outperformed both CID and HCD in terms of number of identified metallated peptide fragments in the mass spectra and the localization of the binding sites. Only ETD allowed the simultaneous and exact determination of Met1 and His68 residues as binding partners for oxaliplatin. CID-MS/MS experiments were carried out on orbitrap and ion cyclotron resonance (ICR)-FT mass spectrometers and both instruments yielded similar results with respect to number of metallated fragments and the localization of the binding sites. A comparison of the protein secondary structure with the intensities of peptide fragments generated by collisional activation of the [Ub + Pt-(chxn)] adduct [chxn = (1R,2R)-cyclohexanediamine] revealed a correlation with cleavages in solution phase random coil areas, indicating that the N-terminal β-hairpin and α-helix structures are retained in the gas phase.     

Conformer-dependent proton-transfer reactions of ubiquitin ions

The conformations of ubiquitin ions before and after being exposed to proton transfer reagents have been studied by using ion mobility/mass spectrometry techniques. Ions were produced by electrospray ionization and exposed to acetone, acetophenone, n-butylamine, and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene. Under the conditions employed, the +4 to +13 charge states were formed and a variety of conformations, which we have characterized as compact, partially folded, and elongated, have been observed. The low charge state ions have cross sections that are similar to those calculated for the crystal conformation. High charge states favor unfolded conformations. The ion mobility distributions recorded after ions have been exposed to each base show that the lowest charge state that is formed during proton-transfer reactions favors a compact conformation. More open conformations are observed for the higher charge states that remain after reaction. The results show that for a given charge state, the apparent gas-phase acidities of the different conformations are ordered as compact < partially folded < elongated.     

Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes

Electrospray ionization (ESI) mass spectrometry (MS) in both the positive and negative ion mode has been used to study protein unfolding transitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of substantially higher charge states than the tightly folded protein in both modes of operation. Surprisingly, the acid-induced unfolding of cyt c as well as the acid and the base-induced unfolding of ubiquitin show different behavior: In these three cases protein unfolding only leads to marginal changes in the negative ion charge state distributions, whereas in the positive ion mode pronounced shifts to higher charge states are observed. This shows that ESI MS in the negative ion mode as a method for probing conformational changes of proteins in solution should be treated with caution. The data presented in this work provide further evidence that the conformation of a protein in solution not its charge state is the predominant factor for determining the ESI charge state distribution in the positive ion mode. Furthermore, these data support the hypothesis of a recent study (Konermann and Douglas, Biochemistry 1997, 36, 12296–12302) which suggested that ESI in the positive ion mode is not sensitive to changes in the secondary structure of proteins but only to changes in the tertiary structure.     

Hydrogen/deuterium exchange in parallel with acid/base induced protein conformational change in electrospray droplets

The exposure of electrospray droplets to vapors of deuterating reagents during droplet desolvation in the interface of a mass spectrometer results in hydrogen/deuterium exchange (HDX) on the sub‐millisecond time scale. Deuterated water is used to label ubiquitin and cytochrome c with minimal effect on the observed charge state distribution (CSD), suggesting that the protein conformation is not being altered. However, the introduction of deuterated versions of various acids (e.g., CD₃COOD and DCl) and bases (ND₃) induces unfolding or refolding of the protein while also labeling these newly formed conformations. The extent of HDX within a protein CSD associated with a particular conformation is essentially constant, whereas the extent of HDX can differ significantly for CSDs associated with different conformations from the same protein. In some cases, multiple HDX distributions can be observed within a given charge state (as is demonstrated with cytochrome c) suggesting that the extent of HDX and CSDs share a degree of complementarity in their sensitivities for protein conformation. The CSD is established late in the evolution of ions in electrospray whereas the HDX process presumably takes place in the bulk of the droplet throughout the electrospray process. Back exchange is also performed in which proteins are prepared in deuterated solvents prior to ionization and exposed to undeuterated vapors to exchange deuteriums for hydrogens. The degree of deuterium uptake is easily controlled by varying the identity and partial pressure of the reagent introduced into the interface. Since the exchange occurs on the sub‐millisecond time scale, the use of deuterated acids or bases allows for transient species to be generated and labeled for subsequent mass analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Electron spin resonance studies of propagating radicals in polymerization. I. Methacrylate propagating radicals

Ferric chloride-photosensitized free-radical initiation was used to generate propagating radicals in polymerization of methacrylic acid (MAA), allyl methacrylate (AMA), methyl methacrylate (MMA), 1,3-butylene dimethacrylate (1,3-BDMA), hydroxypropyl methacrylate (HPMA), lauryl methacrylate (LMA), hexyl methacrylate (HMA), and methacrylamide (MA) in rigid glasses of methanol or acetone at near liquid nitrogen temperatures. The formation and conformational changes of these propagating radicals at different temperatures were studied by electron spin resonance (ESR) spectroscopy. When methanol was the rigid glass, ·CH₂OH radicals were formed initially and were stable below ?160°C. As the temperature of the rigid glass was increased, the ·CH₂OH radicals reacted with monomer to yield propagating radicals. With the exception of the propagating radical for methacrylamide, the propagating radicals of the methacrylates examined initially generated five-line ESR spectra which gradually changed to nine-line spectra, as temperature of the rigid glass was increased. It was concluded that one type of propagating radical was formed in all cases. However, when the temperature of the rigid glass was increased, the single structural conformation that initially allowed one of the methylene hydrogens and methyl group to interact with the unpaired electron to generate only a five-line spectrum was changed to yield a second conformation that allowed interaction to generate an additional four-line spectrum. Finally, a mixture of the propagating radical for methacrylate monomer in two structural conformations was obtained, and an ESR spectrum consisting of nine lines (5 + 4 lines) was generated. In the case of the propagating radical for methacrylamide this change to yield two structural conformations evidently was hindered, so that only an ESR spectrum consisting of five lines was generated.     

Probing interaction of melittin with lipid membranes using liquid secondary ion mass spectrometry

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Molecular modeling of the inhibitory mechanism of copper(II) on aggregation of amyloid β-peptide

Aggregation of amyloid β-peptide (Aβ) into insoluble fibrils is a key pathological event in Alzheimer’s disease (AD). Under certain conditions, Cu(II) exhibits strong inhibitory effect on the Zn(II)-induced aggregation, which occurs significantly even at nearly physiological concentrations of zinc ion in vitro. Cu(II) is considered as a potential factor in the normal brain preventing Aβ from aggregating. The possible mechanism of the inhibitory effect of Cu(II) is investigated for the first time by molecular modeling method. In the mono-ring mode, the Y10 residue promotes typical quasi-helix conformations of Aβ. Specially, [Cu-H13(NTT)-Y10(OH)] complex forms a local 3.0₁₀ helix conformation. In the multi-ring mode, the side chains of Q15 and E11 residues collaborate harmoniously with other chelating ligands producing markedly low energies and quasi-helix conformations. [Cu-3N-Q15(O)-E11(O1)] and [Cu-H13(NTT)-Y10(OH)] complex with quasi-helix conformations may prefer soluble forms in solution. In addition, hydrogen-bond interactions may be the main driving force for Aβ aggregation. All the results will provide helpful clues for an improved understanding of the role of Cu(II) in the pathogenesis of AD and contribute to the development of an “anti-amyloid” therapeutic strategy.     

Direct determination of the primary binding site of cisplatin on cytochrome c by mass spectrometry

Protein—cisplatin interactions lie at the heart of both the effectiveness of cisplatin as a therapeutic agent and side effects associated with cisplatin treatment. Because a greater understanding of the protein—cisplatin interactions at the molecular level can inform the design of cisplatin-like agents for future use, mass spectrometric determination of the binding site of cisplatin on a model protein, cytochrome c, was undertaken in this paper. The monoadduct cytochrome c—Pt(NH₃)₂(H₂O) is found to be the primary adduct produced by the cytochrome c—cisplatin interactions under native conditions. To locate the primary binding site of cisplatin, both free cytochrome c and the cytochrome c adducts underwent trypsin digestion, followed by Fourier transform mass spectrometry (FT-MS) to identify unique fragments in the adduct digest. Four such fragments were found in the adduct digest. Tandem mass spectrometry (MS/MS and MS³ indicates that two fragments are Pt(NH₃)₂(H₂O) bound peptides (Gly56-Glu104 and Asn54-Glu104) with one water associated at the peptide bond Lys79∼Met80, and the other two fragments are heme containing peptides (acety1-Gly1-Lys53 and acety1-Gly1-Lys55). The product-ion spectra of the four fragments reveal that Met65 is the primary binding site of cisplatin on cytochrome c.     

Protein conformation in solution by three-dimensional fluorescence spectrometry

The conformations of bovine serum albumin (USA) and egg albumin (EA) in solution and their conformation changes under different conditions were studied by using three-dimensional fluorescence spectrometry (TDFS) such as three-dimensional fluorescence (TDF) spectra and three-dimensional fluorescence polarization (TDFP) spectra with tryptophan residues in protein molecules as an intrinsic fluorescent probe. The results show that the microenvironment of tryptophan residues of protein molecules in various solutions can be directly indicated and TDFS is an effective tool for studying protein conformation in solution. Meantime, some valuable results were obtained.     

A comparative study of in- and post-source decays of peptide and preformed ions in matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Effective temperature and matrix effect

In-source decay (ISD) and post-source decay (PSD) of a peptide ion ([Y₆ + H]⁺) and a preformed ion (benzyltriphenylphosphonium, BTPP) generated by matrix-assisted laser desorption ionization (MALDI) were investigated with time-of-flight mass spectrometry. α-Cyano-4-hydroxycinammic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were used as matrices. For both ions, ISD yield was unaffected by delay time, indicating rapid termination of ISD. This was taken as evidence for rapid expansion cooling of hot “early” plume formed in MALDI. CHCA was hotter than DHB for [Y₆ + H]⁺ while the matrix effect was insignificant for BTPP. The “early” plume temperature estimated utilizing previous kinetic results was 800–900 K, versus 400–500 K for “late” plume. The results support our previous finding that the temperature of peptide ions interrogated by tandem mass spectrometry was lower than most rough estimates of MALDI temperature.     

Deconvoluted Fourier-transform LNT-IR study of coordination copper(II) compounds with dextran derivatives

Deconvoluted Fourier-transform IR spectra, recorded at room and liquid nitrogen temperature, of polysaccharide dextran and its coordination compounds with the copper(II) ion were analyzed in order to find the most specific spectral peculiarities. This allows one to obtain information about the structure and conformation of these polymer compounds. Different influences on the system of intra-and intermolecular interactions were exhibited by analogs recrystallized from D₂O. The changes in intensity and width of the IR bands in the region 1450–1050 cm⁻¹ were related to changes in conformation and short-range interactions of the dextran. In the synthesized copper(II)-dextran complexes, the presence of water molecules was confirmed. The results of the FTIR spectroscopy study allowed one to suggest a predominant crystalline form of the copper(II)-dextran complexes. The text was submitted by the authors in English.