RESUMEN
Allosteric modulation is a general feature of nicotinic acetylcholine receptors, yet the structural components and movements important for conversions among functional states are not well understood. In this study, we examine the communication between the binding sites for agonist and the modulator morantel (Mor) of neuronal α3ß2 receptors, measuring evoked currents of receptors expressed in Xenopus oocytes with the two-electrode voltage-clamp method. We hypothesized that movement along an interface of ß sheets connecting the agonist and modulator sites is necessary for allosteric modulation. To address this, we created pairs of substituted cysteines that span the cleft formed where the outer ß sheet meets the ß sheet constituting the (-)-face of the α3 subunit; the three pairs were L158C-A179C, L158C-G181C and L158C-K183C. Employing a disulfide trapping approach in which bonds are formed between neighboring cysteines under oxidation conditions, we found that oxidation treatments decreased the amplitude of currents evoked by either the agonist (ACh) or co-applied agonist and modulator (ACh + Mor), by as much as 51%, consistent with the introduced bond decreasing channel efficacy. Reduction treatment increased evoked currents up to 89%. The magnitude of the oxidation effects depended on whether agonists were present during oxidation and on the cysteine pair. Additionally, the cysteine mutations themselves decreased Mor potentiation, implicating these residues in modulation. Our findings suggest that these ß sheets in the α3 subunit move with respect to each other during activation and modulation, and the residues studied highlight the contribution of this intramolecular allosteric pathway to receptor function.
Asunto(s)
Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Colinérgicos/farmacología , Peróxido de Hidrógeno/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Moleculares , Morantel/farmacología , Mutación , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Estructura Secundaria de Proteína , Ratas , Receptores Nicotínicos/genética , Xenopus laevisRESUMEN
Computer simulations of electrospray ionization (ESI) and collision-induced dissociation (CID) experiments were employed to examine the informing power associated with "top-down" proteomics implemented with some commonly used mass analyzers, i.e., the quadrupole ion trap (QIT), the Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICRMS), and the time-of-flight (TOF) mass spectrometer. Using a ratio of the separated (or resolved) peaks to the total number of predicted peaks as a measure of informing power, the ESI-MS simulation of a mixture of proteins showed that the FT-ICRMS exhibited the highest informing power among the three instruments being studied, with the QIT giving the lowest informing power, which was expected from the analysis of the "component capacity" of the three approaches. Also as expected on the basis of resolving elements per component, a dramatic increase in the informing power of the approach was obtained when ion/ion proton-transfer reactions were used to reduce the number of peaks and to minimize overlap between ions of different mass and charge but similar mass-to-charge ratio. With the assumptions made in this study, the informing power of the TOF + ion/ion approach rivaled or even exceeded that of the FT-ICRMS approach, despite significantly lower mass resolution. This result stemmed from both a reduction in the number of peaks and their dispersion over a much wider range of mass-to-charge ratios. Similar results were obtained from the CID simulation, where the informing power of different approaches was evaluated on the basis of the ratio of the number of ions for which a mass could be determined unambiguously to the total number of ions in the spectra.
Asunto(s)
Simulación por Computador , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray , Iones/química , Espectrometría de Masas , Proteínas/análisis , Proteómica/instrumentación , ProtonesRESUMEN
Cation radicals formed via gas-phase electron transfer to multiply protonated polypeptides have been found to react with molecular oxygen. Such cation radicals are of interest within the context of electron transfer dissociation, a phenomenon with high utility for the characterization of peptide and protein primary structures. Most of the cation radicals show the attachment of O(2) under room temperature storage conditions in an electrodynamic ion trap. At higher temperatures and under conditions of collisional activation, the oxygen adduct species lose O(2), HO(*), or HO(2)(*), depending upon the identity of the side chain at the radical site. The fragments containing the C-terminus, the so-called z-ions, which are predominantly radical species, engage in reactions with molecular oxygen. This allows for the facile distinction between z-ions and their complementary even-electron c-ion counterparts. Such a capability has utility in protein identification and characterization via mass spectrometry. Intact electron transfer products also show oxygen attachment. Subsequent activation of such adducts show dissociation behavior very similar to that noted for z-ion adducts. These observations indicate that ion/radical reactions can be used to probe the locations of radical sites in the undissociated electron transfer products as well as distinguish between c- and z-type ions.
Asunto(s)
Péptidos/química , Secuencia de Aminoácidos , Compuestos Azo/química , Cationes/química , Radicales Libres/química , Gases , Espectrometría de Masas , Meliteno/química , Datos de Secuencia Molecular , Neurotensina/química , Nitrobencenos/química , Oxígeno/químicaRESUMEN
A commercial quadrupole/time-of-flight (QqTOF) tandem mass spectrometer has been adapted for ion/ion reaction studies. To enable mutual storage of oppositely charged ions in a linear ion trap, the oscillating quadrupole field of the second quadrupole of the system (Q2) serves to store ions in the radial dimension while auxiliary radio frequency is superposed on the end lenses of Q2 during the reaction period to create barriers in the axial dimension. A pulsed dual electrospray (ESI) source is directly coupled to the instrument interface for the purpose of proton transfer reactions. Singly and doubly charged protein ions as high in mass as 66 kDa are readily formed and observed after proton-transfer reactions. For the modified instrument, the mass resolving power is approximately 8000 for a wide m/z range, and the mass accuracy is approximately 20 ppm for external calibration and approximately 5 ppm for internal calibration after ion/ion reactions. Parallel ion parking is demonstrated with a six-component protein mixture, which shows the potential application of reducing spectral complexity and concentrating certain charge states. The current system has high flexibility with respect to defining MS(n) experiments involving collision-induced dissociation (CID) and ion/ion reactions. Protein precursor and CID product masses can be determined with good accuracy, providing an attractive platform for top-down proteomics. Electron transfer dissociation ion/ion reactions are implemented by using a pulsed nano-ESI/atmospheric pressure chemical ionization dual source for ionization. The reaction between protonated peptide ions and radical anions of 1,3-dinitrobenzene formed exclusively c- and z-type fragment ions.
Asunto(s)
Proteínas/análisis , Proteínas/química , Espectrometría de Masas en Tándem/métodos , Algoritmos , Electrones , Iones , Nanotecnología/métodos , Protones , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
The multiple charging phenomenon resulting from electrospray ionization of proteins, while useful for the ability to make several mass measurements on a single component, can lead to highly complex spectra when mixtures are analyzed, as each component can generate multiple ions of distinct mass-to-charge ratio. Ion/ion proton-transfer reactions can overcome this problem by reduction of all components to the +1 charge state, but this typically requires the ability to extend the mass range of the instrument well beyond that available in most commercial instruments. Furthermore, reduction of protein charge to +1 also results in a reduction in detector response. Here it is shown that application of a relatively high amplitude, low-frequency auxiliary ac signal to the end cap electrodes of a 3-D ion trap during an ion/ion reaction can slow the ion/ion reaction rates of ions over a broad m/z range, in a process termed HALF parallel ion parking. Adjustment of the frequency and amplitude of the applied voltage allows the mass range into which the initial ion signal is moved to be controlled, allowing for the simplification of multicomponent mixtures within a mass range that is more commonly available on commercial systems. In addition to decreasing spectral complexity, this is advantageous for mixtures with low-abundance components, as there is less compromise with detector response than in reduction to the +1 charge state. Preliminary evidence also suggests that the ion collision cross section may play an important role in determining which charge states are most significantly inhibited from further ion/ion reactions under a given set of ion parking conditions.
Asunto(s)
Iones , Proteínas/química , Espectrometría de Masa por Ionización de Electrospray , Electricidad EstáticaRESUMEN
Electron-transfer dissociation allows differentiation of isoaspartic acid and aspartic acid residues using the same c + 57 and z - 57 peaks that were previously observed with electron capture dissociation. These peaks clearly define both the presence and the position of isoaspartic acid residues and they are relatively abundant. The lower resolution of the ion trap instrument makes detection of the aspartic acid residue's diagnostic peak difficult because of interference with side-chain fragment ions from arginine residues, but the aspartic acid residues are still clearly observed in the backbone cleavages and can be inferred from the absence of the isoaspartic acid diagnostic ions.
Asunto(s)
Ácido Aspártico/química , Ácido Isoaspártico/química , Electrones , Indicadores y Reactivos , Espectrometría de Masas , Péptidos/químicaRESUMEN
The ion/ion reactions of several dozen reagent anions with triply protonated cations of the model peptide KGAILKGAILR have been examined to evaluate predictions of a Landau-Zener-based model for the likelihood for electron transfer. Evidence for electron transfer was provided by the appearance of fragment ions unique to electron transfer or electron capture dissociation. Proton transfer and electron transfer are competitive processes for any combination of anionic and cationic reactants. For reagent anions in reactions with protonated peptides, proton transfer is usually significantly more exothermic than electron transfer. If charge transfer occurs at relatively long distances, electron transfer should, therefore, be favored on kinetic grounds because the reactant and product channels cross at greater distances, provided conditions are favorable for electron transfer at the crossing point. The results are consistent with a model based on Landau-Zener theory that indicates both thermodynamic and geometric criteria apply for electron transfer involving polyatomic anions. Both the model and the data suggest that electron affinities associated with the anionic reagents greater than about 60-70 kcal/mol minimize the likelihood that electron transfer will be observed. Provided the electron affinity is not too high, the Franck-Condon factors associated with the anion and its corresponding neutral must not be too low. When one or the other of these criteria is not met, proton transfer tends to occur essentially exclusively. Experiments involving ion/ion attachment products also suggest that a significant barrier exists to the isomerization between chemical complexes that, if formed, lead to either proton transfer or electron transfer.
Asunto(s)
Péptidos/química , Protones , Termodinámica , Electrones , Gases/química , Iones/química , Oxidación-ReducciónRESUMEN
In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (approximately 100%) was observed for small peptides (< or =7 amino acids). For medium-sized peptides composed of 8-11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum.
Asunto(s)
Gases , Péptidos/química , Protones , Temperatura , Secuencia de Aminoácidos , Transporte de Electrón , Iones/química , Datos de Secuencia Molecular , Peso MolecularRESUMEN
Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO2-* in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)-S* and Chain A (or B)-SH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M + 2H]+* ions, which appear to be less kinetically stable than the proton transfer [M + H]+ product. When subjected to collision-induced dissociation (CID), the [M + 2H]+* ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M + 2H]+* ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each.
Asunto(s)
Disulfuros/química , Electrones , Péptidos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Sulfitos/química , Secuencia de Aminoácidos , Animales , Bovinos , Iones/química , Lactalbúmina/química , Datos de Secuencia Molecular , Oxitocina/análogos & derivados , Oxitocina/química , Somatostatina/químicaRESUMEN
Electron-transfer dissociation (ETD) in a tandem mass spectrometer is an analytically useful ion/ion reaction technique for deriving polypeptide sequence information, but its utility can be limited by sequential reactions of the products. Sequential reactions lead to neutralization of some products, as well as to signals from products derived from multiple cleavages that can be difficult to interpret. A method of inhibiting sequential ETD fragmentation in a quadrupole ion trap is demonstrated here for the reaction of a triply protonated peptide with nitrobenzene anions. A tailored waveform (in this case, a filtered noise field) is applied during the ion/ion reaction time to accelerate simultaneously first-generation product ions and thereby inhibit their further reaction. This results in a approximately 50% gain in the relative yield of first-generation products and allows for the conversion of more than 90% of the original parent ions into first-generation products. Gains are expected to be even larger when higher charge-state cations are used, as the rates of sequential reaction become closer to the initial reaction rate.
Asunto(s)
Angiotensina I/análisis , Transporte de Electrón , Iones , Fragmentos de Péptidos/análisis , Péptidos/análisis , Análisis de Secuencia de Proteína/métodos , Angiotensina I/química , Aniones , Nitrobencenos/química , Fragmentos de Péptidos/química , Péptidos/química , Protones , Espectrometría de Masas en Tándem/métodosRESUMEN
Glycosylation is an important post-translational modification. Analysis of glycopeptides is difficult using collision-induced dissociation, as it typically yields only information about the glycan structure, without any peptide sequence information. We demonstrate here how a 3D-quadrupole ion trap, using the complementary techniques of collision induced dissociation (CID) and electron-transfer dissociation (ETD), can be used to elucidate the glycan structure and peptide sequence of the N-glycosylated peptide from a fractionated tryptic digest of the lectin from the coral tree, Erythina cristagalli. CID experiments on the multiply protonated glycopeptide ions yield, almost exclusively, cleavage at glycosidic bonds, with little peptide backbone fragmentation. ETD reactions of the triply charged glycopeptide cations with either sulfur dioxide or nitrobenzene anions yield cleavage of the peptide backbone with no loss of the glycan structure. These results show that a 3D-quadrupole ion trap can be used to provide glycopeptide amino acid sequence information as well as information about the glycan structure.
Asunto(s)
Glicopéptidos/química , Transporte de Electrón , Fabaceae/química , Conformación ProteicaRESUMEN
Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.
Asunto(s)
Espectrometría de Masas/métodos , Péptidos/química , Dióxido de Azufre/química , Transporte de Electrón , ProtonesRESUMEN
Gas-phase reactions between multiply charged positive and negative protein ions are carried out in a quadrupole ion trap mass spectrometer. The ions react with one another by proton transfer and complex formation. Proton transfer products and complexes are formed via competitive processes in single ion/ion encounters. The relative contributions of proton transfer versus complex formation are dependent upon the charges of the ions as well as other characteristics of the ions yet to be clearly delineated. No fragmentation of covalent bonds of the protein reactants is observed. A model that considers the trajectories associated with ion/ion interactions appears to hold the most promise in accounting for the results. The formation of bound ion/ion orbits appears to play an important role in determining overall reaction kinetics as well as the distribution of ion/ion reaction products. Tandem mass spectrometry is used to compare protein complexes formed in the gas-phase with those formed initially in solution and subsequently liberated by electrospray; it is shown that both forms of complex dissociate similarly, but the complexes formed in the gas phase can retain a "memory" of their method of formation.
Asunto(s)
Grupo Citocromo c/química , Ubiquitina/química , Animales , Bovinos , Dimerización , Caballos , Iones , Cinética , Protones , Espectrometría de Masa por Ionización de Electrospray/métodos , Vacio , VolatilizaciónRESUMEN
Novel instrumentation has been developed which allows for the sequential injection and subsequent reaction of oppositely-charged ions generated via electrospray ionization (ESI) in a quadrupole ion trap mass spectrometer. The instrument uses a DC turning quadrupole to sequentially direct the two ion polarities into the ion trap from ESI sources which are situated 90 degrees from the axial (z) dimension of the trap, and 180 degrees from one another. This arrangement significantly expands the range of ionic reactants amenable to study over previously-used instrumentation. For example, ion/ion reactions of multiply-charged positive ions with multiply-charged negative ions can be studied. Also, reactions of multiply-charged ions with singly-charged ions of opposite polarity that could not be generated by previously used ionization methods, or that could not be efficiently injected through the ion trap ring electrode, can be studied with the new instrument. This capability allows, for example, the charge state manipulation of negatively-charged precursor and product ions derived from proteins and oligonucleotides via proton transfer reactions with singly-charged cations generated by ESI.
Asunto(s)
Espectrometría de Masa por Ionización de Electrospray/instrumentación , Animales , Aniones , Cationes , Bovinos , Grupo Citocromo c/química , Electrodos , Indicadores y Reactivos , Ubiquitina/químicaRESUMEN
An instrument for the study of gas-phase ion/ion reactions in which three independent sources of ions, namely, two electrospray ionization sources and one atmospheric sampling glow discharge ionization source, are interfaced to a quadrupole ion trap mass analyzer is described. This instrument expands the scope of gas-phase ion/ion reaction studies by allowing for manipulation of the charge states of multiply charged reactant and product ions. Examples are provided involving the formation of protein-protein complexes in the gas phase. Complexes with charge states that cannot be formed from reactant ion charge states present in the normal electrospray charge state distributions can be formed in the new apparatus. Strategies that rely on both reactant ion charge state manipulation and product ion charge state manipulation are demonstrated. In addition, simplification of product ion spectra generated from dissociation of complexes formed via ion/ion reactions can be effected by using the discharge source to reduce the charge state of the product ions to primarily 1+.
Asunto(s)
Gases/análisis , Gases/química , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Animales , Bovinos , Grupo Citocromo c/química , Iones/química , Datos de Secuencia Molecular , Platino (Metal)/química , Ubiquitina/químicaRESUMEN
Ion trap collisional activation of whole protein anions that contain disulfide bonds results in the cleavage of one of the bonds that comprises the disulfide linkage. The disulfide linkage can break at any of three possible locations, giving rise to several products with different partitioning of sulfur atoms. A facile second-generation dissociation occurs at the polypeptide backbone from products formed from cleavage of the nearest C-S bond of a disulfide linkage. This cleavage occurs exclusively at the N-terminal side of the cysteine residue, from which the C-S bond was cleaved, thereby yielding c and z-S type product ions. This secondary reaction is apparently a relatively low-energy reaction with relatively high entropy requirements because it is not observed to be a major process under beam-type collisional activation conditions, but is a major process under ion trap collisional activation conditions. The specificity of this cleavage, as well as the ability to distinguish it from other cleavages by the sulfur atom distribution, make it useful for the identification of unknown proteins via database searching. Furthermore, the pattern of disulfide cleavages can be useful in providing information about the location of post-translational modifications. Examples using bovine pancreatic trypsin inhibitor and ribonuclease A and B are given to illustrate these points.