Multiply Charged Species Research Articles

New approaches to stored cluster ions

Ion traps are “wall-less containers” which allow the extended storage of selected species. During the storage various interaction steps may be repeatedly applied. To this end no further hardware has to be added - in contrast to beam experiments. In this progress report two examples of recent developments are presented: the experiments have been performed with metal clusters stored in a Penning (ion cyclotron resonance) trap. A new experimental scheme has been developed which allows precision measurements of the dissociation energies of polyatomic species. It has been triggered by investigations on the delayed photodissociation of stored metal clusters. However, the technique is also readily available for application to a broad variety of different species and it is not even restricted to trapping experiments. The second development is more closely connected with ion storage in Penning traps: by application of an “electron bath” singly charged anionic clusters can be converted into multiply charged species. Subsequently, they are charge selected and investigated with respect to their reaction upon excitation. In particular, preliminary results indicate that dianionic metal clusters emit two electrons upon photoexcitation whereas the singly charged species show dissociation.

Design and use of multi-affinity surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS): a step toward the design of SPR/MS arrays.

The feasibility of multi-affinity ligand surfaces in biomolecular interaction analysis-mass spectrometry (BIA/MS) was explored in this work. Multi-protein affinity surfaces were constructed by utilizing antibodies to beta-2-microglobulin, cystatin C, retinol binding protein, transthyretin, serum amyloid P and C-reactive protein. In the initial experiments, all six antibodies were immobilized on a single site (flow cell) on the sensor chip surface, followed by verification of the surface activity via separate injections of purified proteins. After an injection of diluted human plasma aliquot over the antibodies-derivatized surfaces, and subsequent MALDI-TOF MS analysis, signals representing five out of the six targeted proteins were observed in the mass spectra. Further, to avoid the complexity of the spectra, the six proteins were divided into two groups (according to their molecular weight) and immobilized on two separate surfaces on a single sensor chip, followed by an injection of human plasma aliquot. The resulting mass spectra showed signals from all proteins. Also, the convolution resulting from the multiply charged ion species was eliminated. The ability to create such multi-affinity surfaces indicates that smaller-size ligand areas/spots can be employed in the BIA/MS protein interaction screening experiments, and opens up the possibilities for construction of novel multi-arrayed SPR-MS platforms and methods for high-throughput parallel protein interaction investigations.

The cornucopia of beam applications

Three themes touched upon in this brief review are: a historical perspective of non-semiconductor ion implantation; novel research directions that have evolved from ion beam-based research; and a new way to view the preponderance of techniques to generate beams. Surface modification of materials by ion beams began with ∼100 keV monomer, singly charged ions and over the years expanded to lower energy, higher current, neutral or multiply charged species and higher molecular weights. As a result of the expansion of beam production to very high molecular weight materials, a new way to view the relationship of beams to high technology is discussed.

New example of proline-induced fragmentation in electrospray ionization mass spectrometry of peptides.

The positive ion electrospray ionization (ESI+) mass spectra of peptides usually display only protonated molecules provided that soft ionization conditions are applied (low cone voltage to prevent in-source dissociations). Such ions can be multiply charged depending on the molecular weight of the studied compounds. We have experienced an unexpected behavior during the ESI analysis of a modified peptide of relatively high mass (3079 Da). A specific fragmentation occurred even under soft energetic conditions, leading to a mass spectrum containing multiply charged molecular and fragment ions. The selective rupture involved the amide bond between the glutamic acid and proline residues (E-P sequence). The successive replacement of each amino acid by an alanine residue (positional scanning study) was undertaken to assess which part of the sequence induced such selective and abundant fragmentation on multiply charged species. The succession P-P was evidenced as the minimum unit giving rise to the first peptide bond rupture in the sequence X-P-P. Any acidic amino acid at the X position (X = D, E) favored the fragmentation by an intramolecular interaction. Such proline-induced fragmentation occurring readily in the source differed from the literature data on the specific behavior of proline-containing peptides where bond ruptures occur solely in dissociation conditions.

Tandem mass spectrometry of intact proteins for characterization of biomarkers from Bacillus cereus T spores.

Intact protein biomarkers from Bacillus cereus T spores have been analyzed by high-resolution tandem Fourier transform ion cyclotron resonance mass spectrometry. Two techniques have been applied for excitation of the isolated multiply charged precursor ion species: sustained off-resonance irradiation/collisionally activated dissociation and electron capture dissociation. Fragmentation-derived sequence tags and BLAST sequence similarity proteome database searches allow unequivocal identification of the major biomarker protein with unprecedented specificity. Sequence-specific fragmentation patterns further confirm protein identification. Moreover, methodology combining accurate mass measurements of intact proteins with additional information contained in a proteome database permits tentative assignment of several other protein biomarkers isolated from the B. cereus T spores. We argue that approaches involving tandem MS of protein biomarkers, combined with bioinformatics, can drastically improve the specificity of individual microorganism identification, particularly in complex environments.

Similarity between condensed phase and gas phase chemistry: fragmentation of peptides containing oxidized cysteine residues and its implications for proteomics

Amino acid residues containing thioethers are easily oxidized during protein purification, derivatization, and/or digestion. For instance, oxidation of methionine residues in proteins during SDS-PAGE is commonly observed. Under low energy collision induced dissociation this gives rise to a second series of fragment ion of lower abundance that are shifted by −64 Da when compared to the oxidized methionine-containing fragments. We report here that alkylated cysteine residues can be found in their oxidized form too, indicating that the oxidation of thioethers can occur during and following protein digestion and not only during SDS-PAGE or reduction and alkylation. Collision induced dissociation experiments on the singly- and multiply-charged species reveals that these peptides preferentially undergo elimination reactions that forms a dehydroalanine from the oxidized, alkylated cysteine residue. This contrasts to the less abundant elimination reaction of peptides containing oxidized methionines which cannot form an α,β-unsaturated compound, but parallels the condensed phased chemistry of sulfoxides. The masses of both precursor and product ions are shifted such that these peptides cannot be identified in database searches with current algorithms. Incorporation of this fragmentation pattern is important for the isotope-coded affinity tag approach since this method is based on peptides containing cysteine residues.

An experimental evaluation of cyclic voltammetry of multicharged species at macrodisk electrodes in the absence of added supporting electrolyte.

The reversible reduction of [S2Mo18O62]4- to [S2Mo18O62]5- and [S2Mo18O62]6- at a glassy carbon macrodisk electrode has been studied by cyclic voltammetry in acetonitrile as a function of the concentration of [(C6H13)4N]4[S2Mo18O62] in the absence and presence of [(C6H13)4N]ClO4 as the added supporting electrolyte. Consideration is given to the influence of scan rate, reference-working electrode distance, [(C6H13)4N]4[S2Mo18O62], and electrolyte concentrations. Experimental data confirm theoretical predictions that cyclic voltammetry at a macrodisk electrode is a viable technique for studies of multiply charged electroactive species without added electrolyte, provided the influence of enhanced complexities associated with effects of increased solution resistance, the mass transport contribution from migration, and convection arising from enhanced density gradients are considered. Enhanced density gradients present in the absence of added supporting electrolyte give rise to a more marked dependence of voltammograms on the angle of the electrode and hence lead to significant distortion of wave shapes at low scan rates. The summation of all these obstacles implies that quantitative evaluation of cyclic voltammograms of multiply charged species requires significantly greater care in the absence than in the presence of added supporting electrolyte.

Electrospray ionization of large multiply charged species proceeds via Dole’s charged residue mechanism

The maximum charge z max on a variety of electrospray ions of globular proteins retaining their native structure, as well as naturally compact starburst dendrimers is compared to the Rayleigh charge z R (based on a water sphere with the ion mass). For most published data examined, z max is between 65% and 110% of z R, providing strong support in favor of Dole’s charged residue mechanism, at least for masses from 3.3 kD up to 1.4 MD. Other large but less compact ions from proteins and linear chains of polyethylene glycols (PEGs) have z max values considerably larger than z R, apparently implying that they also form as charged residues, though from non-spherical drops held together by the polymer backbone.

The onset of coulomb explosions in polyatomic molecules

With the development of high intensity femtosecond lasers, the ionisation and dissociation dynamics of molecules has become an area of considerable interest. Using the technique of femtosecond laser mass spectrometry (FLMS), the molecules carbon disulphide, pyrimidine, toluene, cyclohexanone and benzaldehyde are studied with pulse widths of 50 fs in the near infrared (IR) wavelength region (790 nm). Results are presented and contrasted for laser beam intensities around 10(15) and 10(16) W cm(-2). For the lower intensities, the mass spectra yield dominant singly charged parent ions. Additionally, the appearance of doubly charged parent ions is evident for carbon disulphide, toluene and benzaldehyde with envelopes of doubly charged satellite species existing in these local regions. Carbon disulphide also reveals a small triply charged component. Such atomic-like features are thought to be a strong fingerprint of FLMS at these intensities. However, upon increasing the laser intensity to approximately 10(16) W cm(-2), parent ion dominance decreases and the appearance of multiply charged atomic species occurs, particularly carbon. This phenomenon has been attributed to Coulomb explosions in which the fast absorption of many photons may produce transient highly ionised parent species which can subsequently blow apart. Copyright 1999 John Wiley & Sons, Ltd.

Resonant photofragmentation of methanol at the carbon and oxygen K-edge by high-resolution ion-yield spectroscopy

Resonant photofragmentation of core-excited methanol has been studied with high-resolution partial ion-yield spectroscopy near both the carbon and oxygen K-edge. Non-coincident and coincident fragmentation channels were monitored simultaneously. A steep increase in the coincidence yield curves above the ionization threshold, where the normal Auger decay produces doubly charged or multiply charged species which yield two or more charged fragments, has been observed. A relevant difference in resonant structure has been observed while comparing the fragmentation at the carbon K-edge and the oxygen K-edge. Evidence for inter-atomic Auger decay is presented for one particular single channel around the oxygen K-edge.

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection

Methods for the determination of a semi-synthetic cyclic hexapeptide ( I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard ( II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 Å), 150×4.6 mm C 8 analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor→product ion combinations of m/ z 1093.7→1033.6 and 1094.7→1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–1000 ng/ml using ISP, and 2.5–500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.

Large Molecule Characterization Based upon Individual Ion Detection with Electrospray Ionization-FTICR Mass Spectrometry

We report a new method for mass spectrometric measurements of high-molecular-weight species based on the summation of sequential Fourier transform ion cyclotron resonance (FTICR) spectra of individual multiply charged ions. This approach produces statistically useful mass spectra for large multiply charged molecular species formed by electrospray ionization and circumvents conventional limitations upon achievable resolving power and precision for high-molecular-weight species which arise due to Coulombic constraints. For very large molecules with tens to thousands of charges each, the total number of charges required to define the charge-state distribution, and thus provide accurate mass information, greatly exceeds the useful charge capacity of the FTICR cell. As trapped ion populations approach or exceed this capacity, FTICR performance degrades due to large frequency shifts, peak coalescence phenomena, and rapid loss of ion packet coherence, which effectively precludes high-resolution and precision measurements for molecules above ∼80-kDa size for a 7-T magnetic field strength. The present approach is based on the summation of many spectra having moderate populations of individual ions and relies on sensitivity sufficient for individual ion detection. While the number of trapped ions contributing to each mass spectrum may generally be insufficient to define the isotopic or charge-state distributions (and thus produce accurate information on the molecular weight distribution in a conventional fashion), the present data processing and summation approach suppresses the noise component (as well as smaller signals) that would otherwise be problematic. Importantly, this approach circumvents natural limitations for very high molecular weight species due to Coulombic interactions and thus provides a basis for much greater resolution and mass measurement accuracy than otherwise possible. This paper presents the details of this approach and its demonstration for the 66-kDa protein bovine serum albumin (where the conventional approach is also feasible) and discusses important aspects of the data manipulation.

Electrolytic corrosion of a stainless-steel electrospray emitter monitored using an electrospray–photodiode array system

An electrospray–photodiode array (ES-PDA) system provides a useful alternative to ES-MS for the study of the electrolytic reactions that occur at the point of high voltage (or ground) contact in an ES ion source emitter. These electrolytic reactions charge-balance the loss of an excess of ions of one polarity in the charged ES droplets. This ES-PDA approach is particularly useful when the redox products are either neutral or difficult to detect in the gas phase unaltered. The latter is the case for the multiply charged metal ions derived from ES emitter corrosion. In this study, the anodic corrosion of iron in a stainless-steel ES emitter, detected optically as tris(1,10-phenanthroline)iron(II), [Fe(phen)3 ]2+ , was found using the ES-PDA system to account for the majority of the charge-balancing current under the conditions used. The metal ions derived from emitter corrosion may be produced at levels sufficient to cause chemical background problems in ES-MS. Probably because of the relatively high concentration detection limits of ES-MS for multiply charged inorganic species, this problem has not yet been a major issue. One expects further use of ES as a nebulization source for ICP-AES and ICP-MS, because of the much better concentration detection limits of these techniques, to bring this issue to the forefront.

Procedures for tandem mass spectrometry on an ion trap storage/reflectron time-of-flight mass spectrometer.

In this work, we demonstrate tandem mass spectrometry on an ion trap storage-reflectron time-of-flight mass spectrometer (IT/reTOFMS). Ion isolation and activation were achieved by resonant excitation using multi- and single-frequency waveforms generated from an analog circuit. Product-ion spectra of small polypeptides are obtained, which are comparable in fragmentation to those acquired on sector or hybrid mass spectrometers. Several important parameters governing the tandem mass spectrometry process, including the activation tickle voltage, type of collision gas, activation period and cooling period after the fragmentation were optimized using leucine-enkephalin as a model. Although the limited energy deposition from collisional activation in our experiments does not allow efficient fragmentation of large singly charged polypeptides with m/z higher than 1000, the problem may be partially solved by taking advantage of fragmenting the multiply charged ions produced from the electrospray ionization source as demonstrated for a synthetic polypeptide of molecular weight 2782. Compared to the singly charged form, the reduced m/z of multiply charged forms experience a greater trapping force as described by the pseudopotential well-depth model. Increased pseudopotential well-depths for multiply charged species permit the use of greater fragmentation energy at lower RF potentials. These conditions facilitate the fragmentation of large polypeptides, yet are suitable for trapping singly charged fragments. These experiments indicate that the high efficiency associated with ion dissociation and fragment-ion collection in the trap and the storage capability for detection of ions using the non-scanning mode of the IT/ reTOF analyzer may provide an alternative means for acquiring sequence-specific information of polypeptides at low picomol levels of sensitivity.

Femtosecond excitation dynamics of acetone: Dissociation, ionization, and the evolution of multiply charged elemental species

Recent femtosecond pump–probe experiments have suggested that a stepwise dissociative mechanism is operative for acetone excited to Rydberg states and upper regions of the mixed singlet/triplet state. The present work focuses on the excitation of acetone and acetone clusters to the 3d (or perhaps 4s) electronic intermediate state in order to further explore the operative dissociation mechanisms and the effects of solvation (clustering). As reported herein, results from femtosecond pump–probe experiments suggest that the availability of additional vibrational modes in clusters, where internal energy may be dispersed, increases the fraction of acetyl intermediates which remain behind the barrier to dissociation into methyl and CO fragments. At progressively higher laser fluences, multiply charged elemental carbon and oxygen ions abruptly appear. Interestingly, the extent of their formation is observed to depend on both laser intensity and the relative time delay between the pump and probe laser beams responsible for their occurrence.

Small Multiply Charged Anions as Building Blocks in Chemistry

Chemists view most if not all ionic crystals as composed of autonomously stable negative and positive ions. These building block ions can be singly or multiply charged (e.g., NaCl(s) consists of Na+ and Clions, and MgSO4(s) contains Mg2+ and SO4 ions), but they are expected to be stable to the extent that they can be characterized in the laboratory. Dividing crystals, melts, and ionic molecules into positive and negative ions is usually performed via the octet rule. For example, K2SO4 salt is considered to be composed of K+ cations and SO4 anions, within which all constituent atoms possess full octets of valence electrons. Within this point of view, chemists interpret the physical, reactivity, optical, and other properties of crystals, melts, and liquid ionic salts in terms of properties intrinsic to the corresponding positive and negative ions. For example, in NH4Cl(s), one interprets Raman spectra in terms of crystal phonons as well as internal N-H vibrations belonging to the NH4 cation. This widely accepted approach in chemistry is called into question when one recognizes that many multiply charged anions that occur frequently in salts do not exist as autonomously stable species in which the extra electrons occupy valence molecular orbitals. How then can one view the composition of an ionic material such as a salt in terms of building blocks that cannot be isolated and thus fully characterized? It is this difficulty that forms a primary focus of the present Account. It is very difficult to experimentally prove or disprove the electronic instability of isolated multiply charged anions. The absence of corresponding signals in mass spectral data does not necessarily mean that the ions do not exist; it could be that, under the experimental source conditions, these anions are not formed. Alternatively, mass spectral peaks cannot prove stability; the species may be metastable yet long-lived enough to survive to the ion detector. The significant increase in the reliability of electronic structure computer techniques allows the question of the instability of multiply charged anions to be addressed using ab initio methods. Moreover, in recent years, substantial progress has been made in the ab initio study of multiply charged species, specifically. Three very good reviews on theoretical and experimental studies have appeared in the last three years.1,2 However, because the reviews were devoted primarily to studies of electronically stable multiply charged anions, many issues related to unstable anions were not included and thus need to be addressed in this work. When we discuss the stability of multiply charged anions, we consider three types of stability. The first is electronic stability of the anion. If An-, at its own optimal geometry, is more stable than the corresponding A(n-1)at the same geometry, we consider Anto be vertically electronically stable. If Anat its optimal geometry is more stable than A(n-1)at its own optimal geometry, we consider Anto be adiabatically electronically stable. In addition, there is the issue of geometrical stability. If Anhas all real vibrational frequencies at its optimal geometry, it is locally geometrically stable. Finally, if Anis more stable than any possible dissociation fragment, it is thermodynamically stable. Clearly, if an anion is thermodynamically stable, it must also be electronically and geometrically stable. Most small multiply charged anions are not thermodynamically stable, although many are electronically and locally geometrically stable and may have large barriers to dissociation or to autodetachment. Such anions can be studied experimentally because they are long-lived. However, many multiply charged anions that are only vertically electronically stable are very difficult to probe experimentally because of their very short lifetimes.

Peptide nucleic acid characterization by MALDI-TOF mass spectrometry.

Peptide nucleic acids (PNAs) are a new class of DNA mimics in which the regular nucleobases of adenine, thymine, cytosine, and guanine are connected via a peptide-like backbone. PNA molecules retain the same Watson-Crick base pairing as regular oligonucleotides, with the added benefits of greater specificity and resistance to enzymatic digestion. While the use of PNAs has grown rapidly because of their potential applications in biotechnology, little work has been done on developing analytical procedures for characterizing them. We have found matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to be an effective tool for PNA analysis. PNA molecules survive the MALDI process intact and are easily ionized with almost no multiply-charged species. These features allow mixtures to be easily characterized. Traditional protein matrices (e.g., sinapinic acid,2,5-dihydroxybenzoic acid, alpha-cyano-4-hydroxycinnamic acid) were found to be superior to DNA matrices (e.g., trihydroxy-acetophenone, 3-hydroxypicolinic acid, picolinic acid). In addition, the new DNA matrix 6-aza-2-thiothymine worked well. The ability of MALDI-TOF-MS to ascertain PNA purity and sequence information at low picomole levels will be important as greater quality control of PNA synthesis is needed (e.g., when PNAs are used as antisense or antigene drugs).

Mass determination of human immunoglobulin IgM using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Monoclonal human immunoglobulin IgM has been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Using a matrix of sinapinic acid with a laser wavelength of 355 nm, positive ions with 2 to 6 charges were observed in the mass spectra. Two major molecular species were present. The molecular weights estimated from the multiply-charged signals were found to decrease systematically with decrease in the charge state of the ion. This effect was determined to stem from the non-linear influence of the initial kinetic energy of desorption on the final kinetic energy of the multiply-charged species. A method of successive approximations, with the molecular weights and desorption velocities of the analytes as variables, was used to correct for these non-linearities. Mean molecular weights for the two IgM species were found to be 939,000 +/- 2000 and 982,000 +/- 2000 Da. An initial desorption velocity of 565 +/- 10 ms/s was determined for both.

Characterization of a Bernas ion source for multiply charged ion implantation

Due to concerns about energy purity and reduced beam current, the use of multiply charged ions to achieve higher effective ion energies with a fixed acceleration potential has not been common for implantation users in the semiconductor industry. Energy purity is compromised primarily by charge exchange in the implanter beamline, caused by neutral gas originating from the ion source extraction aperture. Beam current has been an issue, since traditional implanter ion sources, such as the Freeman source, produce very limited currents of multiply charged species. At low beam currents, the implanter is not economical to use, hence the lack of commercial use of multiply charged ion implantation. Ion sources that address these issues must also meet requirements for adequate source lifetime, simplicity of operation (for computer control) and maintenance, and low cost of ownership. This paper details beam energy purity and usable beam currents for a new medium current Bernas ion source as compared to a standard Freeman ion source. The results show significant performance improvements, while also increasing the ion source lifetime.

Analysis of aluminium alloys using inductively coupled plasma and glow discharge mass spectrometry

The application of inductively coupled plasma mass spectrometry (ICP-MS) and glow discharge mass spectrometry (GD-MS) to the analysis of aluminium alloys is presented. The spectral characteristics of these two techniques are compared and contrasted. For the most part GD mass spectra are simpler than ICP mass spectra in that species originating from the components of air, water and solution solutes are essentially absent in GD spectra. However, GD spectra show the presence of multiply charged Ar species, and matrix and analyte based argide species are more prevalent than oxide species, the opposite being the case with the ICP. For both techniques, potential spectral interferences are evaluated and matrix effects are illustrated. For ICP-based analyses, it is shown that matrix effects can be minimized by adjustment of the nebulizer gas flow rate and by the use of an internal standard which is easy to add to the dissolved samples. For GD-based analyses, a matrix effect exists between low and high alloy aluminium samples and it is shown that the signal from AlAr can be used as an internal standard to minimize this matrix effect. Finally both techniques were applied to the analysis of a range of Alcan and Alcoa aluminium standards.