Gas-phase ionization/desolvation processes and their effect on protein charge state distribution under matrix-assisted laser desorption/ionization conditions

The charge state distribution of proteins was studied as a function of experimental conditions, to improve the understanding of the matrix-assisted laser desorption/ionization (MALDI) mechanisms. The relative abundances of the multiply-charged ions appear to be a function of the matrix chosen, the laser fluence and the matrix-to-analyte molar ratio. A correlation is found between the matrix proton affinity and the yield of singly- versus multiply-charged ions. These results are in good agreement with a model in which gas-phase intracluster reactions play a significant role in analyte ion formation. A new model for endothermic desolvation processes in ultraviolet/MALDI is presented and discussed. It is based upon the existence of highly-charged precursor clusters and, complementary to the ion survivor model of Karas et al., assumes that two energy-dependent processes exist: (i) a soft desolvation involving consecutive losses of neutral matrix molecules, leading to a multiply-charged analyte and (ii) hard desolvation leading to a low charge state analyte, by consecutive losses of charged matrix molecules. These desolvations pathways are discussed in terms of kinetically limited processes. The efficiency of the two competitive desolvation processes seems related to the internal energy carried away by clusters during ablation.     

Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors

A new model for the ionization processes in UV matrix-assisted laser desorption/ionization (MALDI) which accounts for the major phenomena observed is presented and discussed. The model retains elements of earlier approaches, such as photoionization and photochemical reactions, but it redefines these in the light of new working questions, most importantly why only singly charged ions are detected. Based on experimental evidence, the formation of singly and multiply charged clusters by a deficiency/excess of ions and also by photoionization and subsequent photochemical processes is pointed out to be the major ionization processes, which typically occur in parallel. The generation of electrons and their partial loss into the surrounding vacuum and solid, on the one hand, results in a positively charged ion-neutral plume facilitating a high overall ionization yield. On the other hand, these electrons, and also the large excess of protonated matrix ions in the negative ion mode, induce effective ion reneutralization in the plume. These neutralization processes are most effective for the highly charged cluster ions initially formed. Their fragmentation behaviour is evidenced in fast metastable fragmentation characteristics and agrees well with an electron capture dissociation mechanism and the enthalpy transfer upon neutralization forms the rationale for the prominent fragmentation and intense chemical noise accompanying successful MALDI. Within the course of the paper, cross-correlations with other desorption/ionization techniques and with earlier discussions on their mechanisms are drawn.     

If the ionization mechanism in fast-atom bombardment involves ion/molecule reactions, what are the reagent ions? The time dependence of fast-atom bombardment mass spectra and parallels to chemical ionization

The evaporation in vacuo of the matrices used and the particle-induced desorption of matrix molecules in fast-atom bombardment (FAB) contribute to a proposed high pressure region above the FAB matrix known as the selvedge region. If the neutral number density is sufficiently high, ions formed upon bombardment may undergo collisions with molecules, yielding matrix-related cluster ions and, in cases when the analyte is desorbed in neutral form, protonated and deprotonated analyte molecules. Similarities with the chemical ionization (CI experiment have been pointed out previously and are further developed here. If FAB is similar to CI, then the response depends on the structures of the reagent ions — those ions that react with gas phase analyte molecules. We consider here the time dependence of positive and negative ion FAB spectra to attempt to identify the reagent ions of FAB. A model is suggested for the FAB ion source which evaluates similarities to a CI source, as well as spatial aspects that are unique to desorption/ionization techniques.     

Reduction of organic dyes in matrix-assisted laser desorption/ionization and desorption/ionization on porous silicon

Reduction of analytes in matrix-assisted laser desorption/ionization (MALDI) often obscures the actual determination of molecular structure. To address the redox reactions in laser desorption/ionization processes, the organic dyes Methylene Blue, Janus Green B, Crystal Violet and Rhodamine B were analyzed by MALDI or by desorption/ionization on porous silicon (DIOS). Susceptibility to reduction in MALDI was dependent on both the reduction potentials of analytes and the molar ratio of analyte to matrix molecules. Addition of Cu(II) ions as an electron scavenger suppressed the reduction of Methylene Blue in MALDI. The results suggested that electron transfer to analytes from the sample target and/or from the matrix contributed to the reduction. In DIOS, the reductions of organic dyes were more prominent than in MALDI, and were not prevented by Cu(II) ion doping, probably due to direct contact of the analytes with silicon which had little electric resistance.     

On the role of defects and surface chemistry for surface-assisted laser desorption ionization from silicon

The generation of ions from silicon substrates in surface-assisted laser desorption ionization (SALDI) has been studied using silicon substrates prepared and etched by a variety of different methods. The different substrates were compared with respect to their ability to generate peptide mass spectra using standard liquid sample deposition. The desorption/ionization processes were studied using gas-phase analyte deposition. Mass spectra were obtained from compounds with gas-phase basicities above 850 kJmol and with molecular weights up to 370 Da. UV, VIS, and IR lasers were used for desorption. Ionization efficiencies were measured as a function of laser fluence and accumulated laser irradiance dose. Solvent vapors were added to the ion source and shown to result in fundamental laser-induced chemical and physical changes to the substrate surfaces. It is demonstrated that both the chemical properties of the substrate surface and the presence of a highly disordered structure with a high concentration of "dangling bonds" or deep gap states are required for efficient ion generation. In particular, amorphous silicon is shown to be an excellent SALDI substrate with ionization efficiencies as high as 1%, while hydrogen-passivated amorphous silicon is SALDI inactive. Based on the results, a novel model for SALDI ion generation is proposed with the following reaction steps: (1) the adsorption of neutral analyte molecules on the SALDI surface with formation of a hydrogen bond to surface Si-OH groups, (2) the electronic excitation of the substrate to form free electron/hole pairs (their relaxation results in trapped positive charges in near-surface deep gap states, causing an increase in the acidity of the Si-OH groups and proton transfer to the analyte molecules), and (3) the thermally activated dissociation of the analyte ions from the surface via a "loose" transition state.     

Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

A simple device is described for desolvation of highly charged matrix/analyte clusters produced by laser ablation leading to multiply charged ions that are analyzed by ion mobility spectrometry-mass spectrometry. Thus, for example, highly charged ions of ubiquitin and lysozyme are cleanly separated in the gas phase according to size and mass (shape and molecular weight) as well as charge using Tri-Wave ion mobility technology coupled to mass spectrometry. This contribution confirms the mechanistic argument that desolvation is necessary to produce multiply charged matrix-assisted laser desorption/ionization (MALDI) ions and points to how these ions can be routinely formed on any atmospheric pressure mass spectrometer.     

Peptides investigated by laser desorption—multiphoton ionization mass spectrometry

Laser evaporation of intact neutral molecules into a supersonic beam combined with multiphoton ionization (MUPI) is used to study the fragmentation behaviour of peptides. Owing to the separation of desorption and ionization, an optimization of these processes can be applied to the sample. The investigation of mixtures containing hydrophobic and hydrophilic peptides shows equal probabilities for detection of the two molecules, demonstrating that the neutral yield of both classes of compound is equal in the desorption process. A loss of sensitivity is not observed. By employing the feature of tunable fragmentation, it is possible to sequence peptides in the gas phase. At low laser intensities only the molecular ions are formed. By increasing photon intensities, fragmentation reactions are induced. Owing to the nature of the multiphoton ionization, these mass spectra (at moderate laser powers) contain few and only structurally dependent signals. The molecular ion of the sample investigated is detected in every case.     

Atmospheric pressure laser desorption/chemical ionization mass spectrometry: a new ionization method based on existing themes

We have designed and constructed an atmospheric pressure laser desorption/chemical ionization (AP-LD/CI) source that utilizes a laser pulse to desorb intact neutral molecules, followed by chemical ionization via reagent ions produced by a corona discharge. This source employs a heated capillary atmospheric pressure inlet coupled to a quadrupole ion trap mass spectrometer and allows sampling under normal ambient air conditions. Preliminary results demonstrate that this technique provides approximately 150-fold increase in analyte ions compared to the ion population generated by atmospheric pressure infrared matrix-assisted laser desorption/ionization (AP-IR-MALDI).     

Compatibility of electron ionization and soft ionization methods in gas chromatography/orthogonal time‐of‐flight mass spectrometry

Orthogonal acceleration time‐of‐flight (oa‐TOF) mass spectrometry (MS) was coupled to gas chromatography (GC) to measure ion yields (ratio of ion counts to number of neutrals entering the ion source) and signal‐to‐noise (S/N) in the electron ionization (EI) mode (hard ionization) as well as in the soft ionization modes of chemical ionization (CI), electron capture negative ion chemical ionization (NICI) and field ionization (FI). Mass accuracies of the EI and FI modes were also investigated. Sixteen structurally diverse volatile organic compounds were chosen for this study. The oa‐TOF mass analyzer is highly suited for FI MS and provided an opportunity to compare the sensitivity of this ionization method to the more conventional ionization methods. Compared to the widely used quadrupole mass filter, the oa‐TOF platform offers significantly greater mass accuracy and therefore the possibility of determining the empirical formula of analytes. The findings of this study showed that, for the instrument used, EI generated the most ions with the exception of compounds able to form negative ions readily. Lower ion yields in the FI mode were generally observed but the chromatograms displayed greater S/N and in many cases gave spectra dominated by a molecular ion. Ion counts in CI are limited by the very small apertures required to maintain sufficiently high pressures in the ionization chamber. Mass accuracy for molecular and fragment ions was attainable at close to manufacturer's specifications, thus providing useful information on molecular ions and neutral losses. The data presented also suggests a potentially useful instrumental combination would result if EI and FI spectra could be collected simultaneously or in alternate scans during GC/MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation on the role of pneumatic aspects in electrospray, desorption electrospray surface ionization and surface activated chemical ionization

This review reports the results of some studies carried out by us on the role of pneumatic aspects in electrospray and desorption electrospray surface ionization, with the aim to propose some relevant aspects of the mechanisms involved in these ionization methods. Electrospray ion sources, with the exception of the nano- electrospray source, operate with the concurrent action of a strong electrical field and a supplementary coaxial gas flow. The electrical field is responsible for electrospraying of the analyte solution but the use of a coaxial gas flow leads to a significant increase of the analyte signal and allows the use of higher solution flows. However, by employing capillary voltages much lower than those necessary to activate the electrospray phenomenon, analyte ions are still observed and this indicates that different mechanisms must be operative for ion production. Under these conditions, ion generation could take place from the neutral pneumatically sprayed droplet by field-induced droplet ionization. Also in the case of desorption electrospray ionization (DESI), and without any voltage on the spraying capillary as well as on the surface of interest, ions of analytes present on the surface become detectable and this shows that desorption/ionization of analytes occurs by neutral droplets impinging the surface. Consequently, the pneumatic effect of the impinging droplets plays a relevant role, and for these reasons the method has been called pneumatic assisted desorption (PAD). Some analogies existing between PAD and surface activated chemical ionization (SACI), based on the insertion of a metallic surface inside an atmospheric pressure chemical ionization source operating without corona discharge, are discussed.     

Mass spectra of new heterocycles: XIV. Investigation of 2-(alkyl- and propargylsulfanyl)-3-(ferrocenylmethoxy)pyrroles by electron ionization and laser desorption/ionization

By mass spectrometry methods applying electron ionization (70 eV) and laser desorption/ionization (NALDI, MALDI) 1-(alkyl-, cycloalkyl-, vinyloxyethyl-, and aryl)substituted 2-(alkyl- and propargylsulfanyl)-3-(ferrocenylmethoxy)pyrroles were studied and characterized. At the electron ionization the peak of the molecular ion possesses very low intensity. The main fraction ions are the ferrocenylmethyl ion (the most abundant peak) and its decomposition ions. Under the NALDI conditions molecular ions and protonated molecules [M + H]⁺ are generated. The general fragmentation rules in the field-free region are determined. By an example of 1-phenyl derivative of pyrrole the MALDI procedure was also demonstrated to be suitable for investigation of 2-(alkylsulfanyl)-3-(ferrocenylmethoxy)pyrroles.     

Fe+ chemical ionization of peptides

Laser-desorbed peptide neutral molecules were allowed to react with Fe⁺ in a Fourier transform mass spectrometer, using the technique of laser desorption/chemical ionization. The Fe⁺ ions are formed by laser ablation of a steel target, as well as by dissociative charge-exchange ionization of ferrocene with Ne⁺. Prior to reaction with laser-desorbed peptide molecules, Fe⁺ ions undergo 20–100 thermalizin collisions with xenon to reduce the population of excited-state metal ion species. The Fe⁺ ions that have not experienced thermalizing collisions undergo charge exchange with peptide molecules. Iron ions that undergo thermalizing collisions before they are allowed to react with peptides are found to undergo charge exchange and to form adduct species [M + Fe⁺] and fragment ions that result from the loss of small, stable molecules, such as H₂O, CO, and CO₂, from the metal ion-peptide complex.     

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.     

Factors influencing the analytical performance of an atmospheric sampling glow discharge ionization source as revealed via ionization dynamics modeling

A kinetic model is developed for the dynamic events occurring within an atmospheric sampling glow discharge that affect its performance as an ion source for analytical mass spectrometry. The differential equations incorporate secondary electron generation and thermalization, reagent and analyte ion formation via electron capture and ion-molecule reactions, ion loss via recombination processes, diffusion, and ion-molecule reactions with matrix components, and the sampling and pumping parameters of the source. Because the ion source has a flow-through configuration, the number densities of selected species can be estimated by applying the steady-state assumption. However, understanding of its operation is aided by knowledge of the dynamic behavior, so numerical methods are applied to examine the time dependence of those species as well. As in other plasma ionization sources, the ionization efficiency is essentially determined by the ratio of the relevant ion formation and recombination rates. Although thermal electron and positive reagent ion number densities are comparable, the electron capture/ion-molecule reaction rate coefficient ratio is normally quite large and the ion-electron recombination rate coefficient is about an order of magnitude greater than that for ion-ion recombination. Consequently, the efficiency for negative analyte ion formation via electron capture is generally superior to that for positive analyte ion generation via ion-molecule reaction. However, the efficiency for positive analyte ion formation should be equal to or better than that for negative analyte ions when both ionization processes occur via ion-molecule reaction processes (with comparable rate coefficients), since the negative reagent ion density is considerably less than that for positive reagent ions. Furthermore, the particularly high number densities of thermal electrons and reagent ions leads to a large dynamic range of linear response for the source. Simulation results also suggest that analyte ion number densities might be enhanced by modification of the standard physical and operating parameters of the source.     

Reaction of analyte ions with neutral chemical ionization gas

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Ion-molecule reactions of neutral methane with analyte ions under normal methane chemical ionization conditions are discussed. Reactant ions can be generated by direct electron ionization (EI) fragmentation, chemical ionization (CI) fragmentation, or collision-induced dissociation (CID). Examples in which products of such reactions appear in mass spectra in both conventional CI sources in “beam” instruments and low pressure CI in a quadrupole ion trap are presented. Also shown is an example in which MS/MS product ions react with neutral methane used for CI in an ion trap. It is shown that it is relatively straightforward to recognize such reactions in a quadrupole ion trap and in certain cases to minimize or preclude them. Effects of various operating parameters have been investigated and are discussed.     

About the plausible contribution of field ionization in the mechanism of the formation of dyes of ions under conditions of laser desorption/ionization from a nanostructurized graphite surface

An approach is proposed for the estimation of the contribution of field ionization (FI) to the mechanism of dye ion formation under the conditions of laser desorption/ionization (LDI) from a nanostructurized graphite surface. As test systems, rough graphite layers with dyes, e.g., imidazophenazine derivatives applied to them were chosen; these ensure FI in a strong electric field. The dyes form three neutral precursors upon reduction and various types of ions in different ionization methods. It was found that the mass distribution within the group of peaks formed by the initial dye molecule and the products of its reduction in the positive ion mode upon LDI from a rough graphite surface is shifted to lower masses by one atomic mass unit in comparison to the distribution recorded for LDI from a smooth metal support. The analysis of plausible pathways of ion formation has shown that such a shift may be due to the superposition of ions formed by the FI mechanism on a graphite substrate with a number of ions formed by protonation in LDI with no dependence on the support type. In the negative ion mode, the registration of LDI dye spectra succeeded only if the graphite substrates used favored negative FI and electron emission enhanced by the field.     

Electron ionization of SiCl4

Relative partial ionization cross sections (PICS) for the formation of fragment ions following electron ionization of SiCl(4), in the electron energy range 30-200 eV, have been determined using time-of-flight mass spectrometry coupled with an ion coincidence technique. By this method, the contributions to the yield of each fragment ion from dissociative single, double, and triple ionization, are distinguished. These yields are quantified in the form of relative precursor-specific PICS, which are reported here for the first time for SiCl(4). For the formation of singly charged ionic fragments, the low-energy maxima appearing in the PICS curves are due to contributions from single ionization involving predominantly indirect ionization processes, while contributions to the yields of these ions at higher electron energies are often dominated by dissociative double ionization. Our data, in the reduced form of relative PICS, are shown to be in good agreement with a previous determination of the PICS of SiCl(4). Only for the formation of doubly charged fragment ions are the current relative PICS values lower than those measured in a previous study, although both datasets agree within combined error limits. The relative PICS data presented here include the first quantitative measurements of the formation of Cl(2) (+) fragment ions and of the formation of ion pairs via dissociative double ionization. The peaks appearing in the 2D ion coincidence data are analyzed to provide further information concerning the mechanism and energetics of the charge-separating dissociations of SiCl(4) (2+). The lowest energy dicationic precursor state, leading to SiCl(3) (+) + Cl(+) formation, lies 27.4 ± 0.3 eV above the ground state of SiCl(4) and is in close agreement with a calculated value of the adiabatic double ionization energy (27.3 eV).     

On the use of single-photon ionization for inorganic surface analysis

Summary A general surface analysis method has been developed based on non-selective photoionization of sputtered or desorbed neutral atoms and molecules above the surface, followed by time-of-flight mass spectrometry. The approach, currently utilizes two main types of ionizing radiation and a variety of desorption probes. For photoionization, coherent untuned sources are used; an intense focused pulsed UV laser beam is used for non-resonant multiphoton ionization to give elemental and limited chemical information, usually used for inorganic analysis; a coherent VUV source is used for single-photon ionization at 118 nm (10.5 eV) produced by frequency tripling of 355 nm light from a Nd:YAG laser. This paper focuses on single-photon ionization for inorganic systems. The desorption probes used are ion, electron, and laser beams as well as thermal desorption. For depth profiling, ion beams are specifically used. Any focused desorption probe beam can provide lateral spatial resolution.     

Trapping molecular ions formed via photo-associative ionization of ultracold atoms

The formation of (40)Ca(2)(+) molecular ions is observed in a hybrid (40)Ca magneto-optical and ion trap system. The molecular ion formation process is determined to be photo-associative ionization of ultracold (40)Ca atoms. A lower bound for the two-body rate constant is found to be beta ≥ 2 ± 1 × 10(-15) cm(3) Hz. Ab initio molecular potential curves are calculated for the neutral Ca(2) and ionic Ca(2)(+) molecules and used in a model that identifies the photo-associative ionization pathway. As this technique does not require a separate photo-association laser, it could find use as a simple, robust method for producing ultracold molecular ions.     

A multiplexed post-translational modification monitoring approach on a matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer

Post-translational modifications (PTMs) of proteins are essential for proper function, as they regulate many aspects of a protein's activity and interaction with substrates. When analyzing modified peptides derived from such proteins by mass spectrometry, these modifications can dissociate, producing either a marker ion or neutral loss characteristic of the modification, which have conventionally been monitored with a precursor ion scan or neutral loss scan, respectively. Although powerful, both precursor ion scans and neutral loss scans can only screen for one particular modification at a time. This has led to the development of multiple neutral loss monitoring (MNM) for neutral losses and multiple precursor ion monitoring (MPM) for marker ions on electrospray instruments. Here, we report their implementation on a matrix-assisted laser desorption/ionization (MALDI) instrument as well as the inception of a novel scan strategy termed targeted multiple precursor ion monitoring (tMPM). This latter scan strategy has been developed on a MALDI tandem time-of-flight (TOF/TOF) mass spectrometer for the identification of multiple PTMs via their associated marker ions by manipulating certain components of the instrument, notably the timed ion selector and the delayed extraction source 2. Targeted MPM combined with a second approach, multiple neutral loss monitoring (MNM), is shown to be a successful approach in the identification of PTMs, identifying multiple modified peptides in a complex sample matrix.