Increasing Charge State Research Articles

Increasing fragmentation of disulfide-bonded proteins for top–down mass spectrometry by supercharging

The disulfide bond is an important post-translational modification to form and maintain the native structure and biological functions of proteins. Characterization of disulfide bond linkages is therefore of essential interest in the structural elucidation of proteins. Top–down mass spectrometry (MS) of disulfide-bonded proteins has been hindered by relatively low sequence coverage due to disulfide cross-linking. In this study, we employed top–down ESI–MS with Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD) to study the fragmentation of supercharged proteins with multiple intramolecular disulfide bonds. With charge enhancement upon the addition of sulfolane to the analyte solution, improved protein fragmentation and disulfide bond cleavage efficiency was observed for proteins including bovine β-lactoglobulin, soybean trypsin inhibitor, human proinsulin, and chicken lysozyme. Both the number and relative abundances of product ions representing disulfide cleavage increase with increasing charge states for the proteins studied. Our studies suggest supercharging ESI–MS is a promising tool to aid in the top–down MS analysis of disulfide-bonded proteins, providing potentially useful information for the determination of disulfide bond linkages.

Effect of vacancies on structural, electronic and optical properties of monolayer MoS2: A first-principles study

Effects of vacancies on structural, electronic and optical properties of monolayer MoS2 were investigated using first-principles plane-wave pseudopotential method based on density functional theory. Results show that the band structure and band gap of perfect monolayer MoS2 are in good agreement with the available experimental and theoretical data. Structural analysis indicates that ions surrounding Mo vacancies show an outward relaxation, while that ions surrounding S vacancies exhibit slightly inward relaxation. Electronic analysis implies that the band gaps of defective monolayer MoS2 are smaller than that of perfect one. After introduction of neutral S-vacancy, monolayer MoS2 has changed from direct to indirect band gap. Mo vacancies bring about acceptor-like levels and p-type conductivities, whereas S vacancies lead to donor-like levels and n-type conductivities. With the increasing charge states of vacancies, the band gaps get smaller and the defect energy levels become deeper. Moreover, as the charge states of vacancies increase, the static dielectric constants of monolayer MoS2 with Mo vacancies decrease, whereas the static dielectric constants of monolayer MoS2 with S vacancies increase.

Effect of source tuning parameters on the plasma potential of heavy ions in the 18 GHz high temperature superconducting electron cyclotron resonance ion source

Plasma potentials for various heavy ions have been measured using the retarding field technique in the 18 GHz high temperature superconducting ECR ion source, PKDELIS [C. Bieth, S. Kantas, P. Sortais, D. Kanjilal, G. Rodrigues, S. Milward, S. Harrison, and R. McMahon, Nucl. Instrum. Methods B 235, 498 (2005); D. Kanjilal, G. Rodrigues, P. Kumar, A. Mandal, A. Roy, C. Bieth, S. Kantas, and P. Sortais, Rev. Sci. Instrum. 77, 03A317 (2006)]. The ion beam extracted from the source is decelerated close to the location of a mesh which is polarized to the source potential and beams having different plasma potentials are measured on a Faraday cup located downstream of the mesh. The influence of various source parameters, viz., RF power, gas pressure, magnetic field, negative dc bias, and gas mixing on the plasma potential is studied. The study helped to find an upper limit of the energy spread of the heavy ions, which can influence the design of the longitudinal optics of the high current injector being developed at the Inter University Accelerator Centre. It is observed that the plasma potentials are decreasing for increasing charge states and a mass effect is clearly observed for the ions with similar operating gas pressures. In the case of gas mixing, it is observed that the plasma potential minimizes at an optimum value of the gas pressure of the mixing gas and the mean charge state maximizes at this value. Details of the measurements carried out as a function of various source parameters and its impact on the longitudinal optics are presented.

Investigating the Role of Adducts in Protein Supercharging with Sulfolane

The supercharging effect of sulfolane on cytochrome c (cyt c) during electrospray ionization mass spectrometry (ESI-MS) in the absence of conformational effects was investigated. The addition of sulfolane on the order of 1mM or greater to denaturing solutions of cyt c results in supercharging independent of protein concentration over the range of 0.1 to 10μM. While supercharging was observed in the positive mode, no change in the charge state distribution was observed in the negative mode, ruling out polarity-independent factors such as conformational changes or surface tension effects. A series of sulfolane adducts observed with increasing intensity concurrent with increasing charge state suggests that a direct interaction between sulfolane and the charged sites of cyt c plays an important role in supercharging. We propose that charge delocalization occurring through large-scale dipole reordering of the highly polar supercharging reagent reduces the electrostatic barrier for proximal charging along the cyt c amino acid chain. Supporting this claim, supercharging was shown to increase with increasing dipole moment for several supercharging reagents structurally related to sulfolane.

Sequence Analysis of Peptide:Oligonucleotide Heteroconjugates by Electron Capture Dissociation and Electron Transfer Dissociation

Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.

Modifying the endohedral La-ion location in both neutral and positively charged polyhydroxylated metallofullerenes

We present extensive ab initio density-functional theory calculations in order to analyze the structure and energetics of both neutral and positively charged highly hydroxylated $\text{La}@{\text{C}}_{60}(\text{OH})_{32}{}^{+q}$ and $\text{La}@{\text{C}}_{82}(\text{OH})_{20}{}^{+q}$ ($q=0$, 1, 2, 4, 6, and 8) metallofullerenes. Interestingly we obtain, for the neutral hydroxylated structures, that the location of the encapsulated La atom strongly depends on the precise distribution of the OH groups on the carbon surface. Actually, we found atomic configurations in which the La ion is attached to different regions of the inner surface as well as atomic arrays in which weakly bonded endohedral species are obtained, being located near the center of the structure. The previous strong variations in the endohedral position of the lanthanum atom leads to sizable modifications in its valence state as well as to notable changes in the electronic spectra and in the lowest-energy spin configuration of the molecules, which are all facts that could be used to better identify the atomic structure of these highly relevant metallofullerenes. The positive charging (by means of simply electron detachment) of our here-considered $\text{La}@{\text{C}}_{60}{(\text{OH})}_{32}$ compounds reveals that the rupture of various C-C bonds of the carbon network can be achieved. However, even if the previous bond breakage leads to the formation of sizable holes in the ${\text{C}}_{60}$ cavity (made of nine-membered rings), we obtain that the encapsulated La atom was unable to escape from the cage. In contrast, in the polyhydroxylated $\text{La}@{\text{C}}_{82}{(\text{OH})}_{20}$ fullerene, no C-C bond breakage is induced with increasing charge state of the cage, defining thus a more stable molecular compound. The previous results are expected to be of fundamental importance in medical and biological applications where the permanent encapsulation of these highly toxic atomic species is required.

Observation of Nano-Dots on HOPG Surface Induced by Highly Charged Arq+ Impact

Highly charged ions (HCIs) have huge potential energy due to their high charge state. When a HCI reaches a solid surface, its potential energy is released immediately on the surface to cause a nano-scale defect. Thus, HCIs are expected to be useful for solid-surface modifications on the nano-scale. We investigate the defects on a highly oriented pyrolytic graphite (HOPG) surface induced by slow highly charged Arq+ ions with impact energy of 20–2000qeV with scanning probe microscopy (SPM). In order to clarify the role of kinetic and potential energies in surface modification, the nano-defects are characterized in lateral size and height corresponding to the kinetic energy and charge state of the HCIs. Both the potential energy and kinetic energy of the ions may influence the size of nano-defect. Since potential energy increases dramatically with increasing charge state, the potential energy effect is expected to be much larger than the kinetic energy effect in the case of extremely high charge states. This implies that pure surface modification on the nano-scale could be carried out by slow highly charged ions. The mean size of nano-defect region could also be controlled by selecting the charge state and kinetic energy of HCI.

The Measurements of the Secondary Ion Emissions on the Charge State of Fast Ions and Clusters

A sample for studying secondary ion emissions was made by pressing powders of carbon nanotubes with purity of 98%. The emission yields of different species of atoms/molecules are measured by using a conventional time of flight (TOF) technique under bombardments of C + q , Si + q ( q =1–6), and Ni + q ( q =1–5), C 2 + q , Si 2 + q ( q =1,3,5), and Ni 2 + q ( q =3,5), C 3 + q , Si 3 + q , and Ni 3 + q ( q =2,4) with energy of 1.5 MeV per atom, respectively. With increasing charge states of the projectiles, the secondary ion emission yields per projectile increase and q 3 -dependence are observed. The size and the charge state of the clusters are larger, the secondary ion emissions are more enhanced. The electronic process plays an important role in secondary ion emissions under fast projectile bombardments.

Which Electrospray-Based Ionization Method Best Reflects Protein-Ligand Interactions Found in Solution? A Comparison of ESI, nanoESI, and ESSI for the Determination of Dissociation Constants with Mass Spectrometry

We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (KD) of the model system hen egg white lysozyme (HEWL) binding to N,N',N''-triacetylchitotriose (NAG3). The best KD value compared with solution data were found for ESSI, 19.4 +/- 3.6 microM. Then, we determined the KDs of the two nucleotide binding sites of adenylate kinase (AK), where we obtained KDs of 2.2 +/- 0.8 microM for the first and 19.5 +/- 8.0 microM for the second binding site using ESSI. We found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the KD for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found KD values with a strong charge state dependence, which were in no case close to literature values for solution phase.

Resolving Oligomers from Fully Grown Polymers with IMS−MS

Ion mobility and mass spectrometry techniques, combined with electrospray ionization, have been used to examine distributions of poly(ethylene glycols) (PEG) with average molecular masses of 6550 and 17900 Da. The analysis provides information about the polymer size distributions as well as smaller oligomers existing over a wide range of charge states and sizes (i.e., [HO(CH2CH2O)xH + nCs]n+, where x ranges from 21 to 151 and n = 2 to 11 for the 6550 Da sample; and, x ranges from 21 to 362 and n = 2 to 23 for the 17 900 Da sample). The present data show that oligomer distributions also fall into families, corresponding to much narrower size distributions for individual charge states; this dramatically simplifies data analysis. For example, we show evidence for baseline resolution of the +10 charge state of polymers. Unlike the charge-state trends reported previously for peptide ion families, which show generally increasing mobilities with increasing charge state (for a given m/z value), the mobilities of [HO(CH2CH2O)xH + nCs]n+ families generally decrease with increasing charge state. This requires that the addition of charges leads to substantial changes in the average structures of the ions. Comparisons of cross section calculations from molecular modeling results for multiply cesiated PEG ions with experimental cross sections indicate that these ions adopt highly extended (in many cases nearly linear) conformations, except for the high degree of coordination of the charged sites.

Protein Desolvation in UV Matrix-Assisted Laser Desorption/Ionization (MALDI)

We describe experiments in MALDI-TOF and MALDI-TOF-TOF showing that the ejection of protein-matrix cluster ions and their partial decay in the source occur in MALDI. The use of radial beam deflection and small size detector in linear mode allows detection of ions with higher time-of-flight and kinetic energy deficit. MALDI-TOF-TOF experiments were carried out by selecting chemical noise ions at m/z higher than that of a free peptide ion. Whatever the selected m/z (up to m/z 300) the molecular peptide ion appeared as the main fragment. The production of protein-matrix clusters and their partial decay in the source was found to increase with the size of the protein (MW from 1000 to 150,000 u), although it decreases with increasing charge state. These effects were observed for different matrices (HCCA and SA) and in a large laser fluence range. Experimental results and calculation highlight that a continuous decay of protein-matrix cluster ions occurs in the source. This decay-desolvation process can account for the high-mass tailing and peak shifting as well as the strong noise/background in the mass spectra of proteins.

Channels of Potential Energy Dissipation during Multiply Charged Argon-Ion Bombardment of Copper

The dissipation of potential energy of multiply charged Ar ions incident on Cu has been studied by complementary electron spectroscopy and calorimetry at charge states between 2 and 10 and kinetic energies between 100 eV and 1 keV. The emitted and deposited fractions of potential energy increase at increasing charge state, showing a significant jump for charge states q>8 due to the presence of L-shell vacancies in the ion. Both fractions balance the total potential energy, thus rendering former hypotheses of a significant deficit of potential energy obsolete. The experimental data are reproduced by computer simulations based on the extended dynamic classical-over-the-barrier model.

Electron yields from uranium dioxide under impact of slow Xe ions

Measurements of the secondary electron yields from uranium dioxide under impact of Xeq+ (q=10–25, 0.03keV⩽EKin⩽81keV) ions are presented. A strong increase of the secondary electron yield is observed with increasing charge state of the projectile both at normal and oblique incidences. No variation of the electron yield with the projectile kinetic energy is observed at normal incidence. Therefore, the electron emission process in highly charged ion – uranium dioxide (UO2) interactions is dominated by potential electron emission.

Influence of Coulombic Repulsion on the Dissociation Pathways and Energetics of Multiprotein Complexes in the Gas Phase

Thermal dissociation experiments, implemented with blackbody infrared radiative dissociation and Fourier-transform ion cyclotron resonance mass spectrometry, are performed on gaseous protonated and deprotonated ions of the homopentameric B subunits of Shiga toxin 1 (Stx1 B5) and Shiga toxin 2 (Stx2 B5) and the homotetramer streptavidin (S4). Dissociation of the gaseous, multisubunit complexes proceeds predominantly by the loss of a single subunit. Notably, the fractional partitioning of charge between the product ions, i.e., the leaving subunit and the resulting multimer, for a given complex is, within error, constant over the range of charge states investigated. The Arrhenius activation parameters (E(a), A) measured for the loss of subunit decrease with increasing charge state of the complex. However, the parameters for the protonated and deprotonated ions, with the same number of charges, are indistinguishable. The influence of the complex charge state on the dissociation pathways and the magnitude of the dissociation E(a) are modeled theoretically with the discrete charge droplet model (DCDM) and the protein structure model (PSM), wherein the structure of the subunits is considered. Importantly, the major subunit charge states observed experimentally for the Stx1 B5(n+/-) ions correspond to the minimum energy charge distribution predicted by DCDM and PSM assuming a late dissociative transition-state (TS); while for structurally-related Stx2 B5(n+) ions, the experimental charge distribution corresponds to an early TS. It is proposed that the lateness of the TS is related, in part, to the degree of unfolding of the leaving subunit, with Stx1 B being more unfolded than Stx2 B. PSM, incorporating significant subunit unfolding is necessary to account for the product ions observed for the S4(n+) ions. The contribution of Coulombic repulsion to the dissociation E(a) is quantified and the intrinsic activation energy is estimated for the first time.

Cluster-assisted generation of multi-charged ions in nanosecond laser ionization of pulsed hydrogen sulfide beam at 1064 and 532 nm

The multi-charged sulfur ions of Sq+ (q? 6) have been generated when hydrogen sulfide cluster beams are irradiated by a nanosecond laser of 1064 and 532?nm with an intensity of 1010~ 1012W.cm?2. S6+ is the dominant multi-charged species at 1064?nm, while S4+, S3+ and S2+ ions are the main multi-charged species at 532?nm. A three-step model (i.e., multiphoton ionization triggering, inverse bremsstrahlung heating, electron collision ionizing) is proposed to explain the generation of these multi-charged ions at the laser intensity stated above. The high ionization level of the clusters and the increasing charge state of the ion products with increasing laser wavelength are supposed mainly due to the rate-limiting step, i.e., electron heating by absorption energy from the laser field via inverse bremsstrahlung, which is proportional to ?2, ? being the laser wavelength.

Single, double and triple ionization of tetraphenyl iron(III) porphyrin chloride

Tetraphenyl iron(III) porphyrin chloride (FeTPPCl) cations are generated in the gas phase by electron impact ionization. The ionization is accompanied by extensive fragmentation as well as formation of doubly and triply charged ions. The most prominent fragments are analyzed and identified by fitting with calculated natural isotope patterns. Appearance energies of the most abundant singly and doubly charged product ions are determined. For the singly charged parent ions FeTPPCl +, CuTPP + and the fragment ion FeTPP + we obtain a value of 9.7 ± 0.5 eV which is about 3 eV higher than the value published for photo ionization of FeTPPCl. The appearance energy of the doubly charged ion FeTPP 2+ is obtained to be 18 eV. The additional loss of one or two phenyl groups requires between 10 and 14 eV more for singly and doubly charged ions. Also, the metastable decay of singly and doubly charged ions is investigated with the mass analyzed ion kinetic energy (MIKE) scan technique, performed on a three sector field mass spectrometer (BEE-geometry). In the mass spectrum and the MIKE scans a strongly reduced stability of the porphyrin ions is observed with increasing charge state.

Momentum-imaging spectroscopy of secondary ions from GaN and SiC surfaces collided with highly charged ions at grazing angle

Three-dimensional momentum of sputtered ions were measured in coincidence with scattered Ar atoms in collisions between Ar q+ ( q = 3, 8, 11, and 14) and 6H-SiC and GaN surfaces at grazing-incidence angle (<0.5°). H +, H 2 +, Si + and Ga + ions were observed in time-of-flight-mass spectra. Sputtering yields of H + and H 2 + increased with increasing charge states of incident ions in proportion to q 4. The momenta of these secondary ions emitted from the surfaces were intensively distributed to the direction of surface normal in spite of the grazing collisions. Almost no significant change among different q was found in the kinetic energy distributions of these secondary ions.

A Fast Flow Tube Study of Gas Phase H/D Exchange of Multiply Protonated Ubiquitin

An electrospray ionization (ESI)/fast-flow technique has been applied to the study of gas phase hydrogen/deuterium (H/D) exchange kinetics. Multiply charged ubiquitin ions [ubiquitin + nH](n)(+), in charge states n = 7-13, were reacted with ND(3). The behavior of ND(3) as exchange reagent is different from that of the previously studied reagents, D(2)O and CH(3)OD. Contrary to those, the maximum number of exchanged hydrogen atoms and the overall exchange rate were observed to increase with increasing charge state of the ubiquitin ions. The results are reagent-dependent because the exchange mechanisms are different for the different reagents. This observation is in agreement with a recent conclusion by Beauchamp and co-workers that contrary to the assumption often expressed in earlier studies, H/D exchange kinetics may not directly reflect ion structures. The results for all three reagents are, however, consistent with observations of previous ion mobility experiments that with increasing charge state the conformers change from more compact, partially folded structures to elongated nearly linear ones. H/D exchange of (ubiquitin + 13H)(13+) with ND(3) leads to two separated ion populations reflecting the possible existence of two conformers with different exchange rates. The ions (ubiquitin + 8H)(8+) and (ubiquitin + 11H)(11+) represent a partially folded structure and an unfolded structure, respectively, and were studied in greater detail. The relative abundances of ions were measured in steps of 0.5 m/z (mass-to-charge ratio), as a function of the ND(3) flow rate. The experimental results were simulated by computer fitted curves based on a recently developed algorithm. The algorithm allows the extraction of sets of grouped rate constants. Eight rate constant groups were deduced for each of the two ions. These rate constants correspond to 32 and 44 H/D exchanges for the 8+ and 11+ charged ions, respectively. The results indicate higher individual rates for most of the exchanged atoms in the 11+ ion compared to the 8+ ion.

Multiple photoionization following 3d5/2-shell threshold ionization of Xe

Multiple photoionization of Xe near the 3d5/2-shell threshold photoionization region is studied by threshold electron–ion coincidence spectroscopy. The coincidence spectra of Xe3+ to Xe7+ ions exhibit characteristic profiles associated with multi-step post-collision interactions in Auger cascades following 3d5/2-shell threshold photoionization. The Auger cascade decay channels leading to the formation of multiply charged ions are deduced from the energies of the profile peaks, which increase gradually with increasing charge state. The formation of Xe3+ to Xe5+ ions is found to arise from cascades of normal Auger decays, whereas the formation of Xe6+ and Xe7+ ions involves double Auger decays. The branching ratio of double to normal Auger decays is estimated to be 0.25 (±0.1) for the decays following the creation of 3d5/2-hole states in Xe.

Electron-impact single and multiple ionization of praseodymium ions

Using the dynamic crossed-beams method, the following absolute cross sections σq,q+n for single (n=1), double (n=2) and triple (n=3) ionization by electron impact have been measured for the reaction e−+Prq+→Pr(q+n)++(n+1)e−n=1:σ2,3σ3,4σ4,5σ6,7σ7,8σ8,9σ11,12n=2:σ1,3σ2,4σ3,5σ4,6n=3:σ1,4σ2,5σ3,6The measurements have been performed in an energy range from the respective threshold up to 1 keV. For the single-ionization cross sections, contributions from excitation–autoionization processes increase with increasing charge state of the parent ion. As no theoretical calculations on praseodymium ions exist, these cross sections are compared to the semi-empirical Lotz formula [Zeitschrift für Physik 232 (1970) 101]. Where contributions of indirect inner-shell processes to the cross section are expected, we conducted energy-scan measurements with high energy resolution. For multiple ionization, no theoretical calculations on praseodymium ions exist. Therefore, the measured cross sections were compared to the semi-empirical formulae by Bélenger et al. [J. Phys. B: At. Mol. Opt. Phys. 30 (1997) 2667] and Fisher et al. [J. Phys. B: At. Mol. Opt. Phys. 28 (1995) 3027].