Kinetic energy measurements of molecular ions ejected into an electric field by matrix-assisted laser desorption.

Measurements of kinetic energy distributions of molecular ions ejected into an extraction field by matrix-assisted laser desorption are reported. The measurements were made in a time-of-flight mass spectrometer with an electrostatic mirror by measuring the reflected signal as a function of the difference between the accelerating voltage and the voltage applied to the mirror. The molecular ions were found to have less kinetic energy than the extraction field alone would normally provide, i.e., we observed an energy deficit. Under conditions typical for a matrix-assisted laser desorption experiment, the deficit is about 24 eV for molecular ions of insulin. The size of the deficit increases with the intensity of the molecular ion signal, and the molecular weight of the protein; it is also larger for negative molecular ions than for positive molecular ions.     

A constant-momentum/energy-selector time-of-flight mass spectrometer

A matrix assisted laser desorption/ionization time-of-flight mass spectrometer has been built with an ion source that can be operated in either constant-energy or constant-momentum acceleration modes. A decreasing electric field distribution in the ion-accelerating region makes it possible to direct ions onto a space-focal plane in either modes of operation. Ions produced in the constant-momentum mode have velocities and, thus, flight times that are linearly dependent on mass and kinetic energies that are inversely dependent on mass. The linear mass dispersion doubles mass resolving power of ions accelerated with space-focusing conditions in constant-momentum mode. The mass-dependent kinetic energy is exploited to disperse ions according to mass in a simple kinetic energy filter constructed from two closely spaced, oblique ion reflectors. Focusing velocity of ions of the same mass can substantially improve ion selection for subsequent post source decay or tandem time-of-flight analyses.     

Intensity dependence of cation kinetic energies from 2,5-dihydroxybenzoic acid near the infrared matrix-assisted laser desorption/ionization threshold

The mechanisms responsible for matrix-assisted laser desorption/ionization (MALDI) are far from being well understood, particularly where infrared laser irradiation is used to initiate the process. We measured the emission yields and kinetic energy distributions of positive ions emitted from 2,5-dihydroxybenzoic acid loaded with angiotensin II in a standard MALDI preparation during irradiation with an infrared free-electron laser tuned to 2.94 microm. As the laser intensity is scanned through the MALDI threshold, we see a marked change in the energy distributions of the matrix ion. Above threshold, the energy distributions of both analyte and matrix cations are constant over a broad range of laser intensities. This behavior does not appear to be consistent with any extant model of the MALDI mechanism.     

Investigating ion-surface collisions with a niobium superconducting tunnel junction detector in a time-of-flight mass spectrometer

The performance of an energy sensitive, niobium superconducting tunnel junction (STJ) detector is investigated by measuring the pulse height produced by impacting molecular and atomic ions at different kinetic energies. Ions are produced by laser desorption and matrix-assisted laser desorption in a time-of-flight mass spectrometer. Our results show that the STJ detector pulse height decreases for increasing molecular ion mass, passes through a minimum at around 2000 Da, and then increases with increasing mass of molecular ions above 2000 Da. The detector does not show a decline in sensitivity for high mass ions as is observed with microchannel plate ion detectors. These detector plus height measurements are discussed in terms of several physical mechanisms involved in an ion-surface collision.     

Degree of Ionization in MALDI of Peptides: Thermal Explanation for the Gas-Phase Ion Formation

Degree of ionization (DI) in matrix-assisted laser desorption ionization (MALDI) was measured for five peptides using α-cyano-4-hydroxycinnanmic acid (CHCA) as the matrix. DIs were low 10(-4) for peptides and 10(-7) for CHCA. Total number of ions (i.e., peptide plus matrix) was the same regardless of peptides and their concentration, setting the number of gas-phase ions generated from a pure matrix as the upper limit to that of peptide ions. Positively charged cluster ions were too weak to support the ion formation via such ions. The total number of gas-phase ions generated by MALDI, and that from pure CHCA, was unaffected by the laser pulse energy, invalidating laser-induced ionization of matrix molecules as the mechanism for the primary ion formation. Instead, the excitation of matrix by laser is simply a way of supplying thermal energy to the sample. Accepting strong Coulomb attraction felt by cations in a solid sample, we propose three hypotheses for gas-phase peptide ion formation. In Hypothesis 1, they originate from the dielectrically screened peptide ions in the sample. In Hypothesis 2, the preformed peptide ions are released as part of neutral ion pairs, which generate gas-phase peptide ions via reaction with matrix-derived cations. In Hypothesis 3, neutral peptides released by ablation get protonated via reaction with matrix-derived cations.     

Application of matrix-assisted laser desorption/ionization to on-line aerosol time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained from single biological aerosol particles using an aerosol time-of-flight mass spectrometer (ATOFMS). The inlet to the ATOFMS was coupled with an evaporation/condensation flow cell that allowed the aerosol to be coated with matrix material as the sampled stream entered the spectrometer. Mass spectra were generated from aerosol composed either of gramicidin-S or erythromycin, two small biological molecules, or from aerosolised spores of Bacillus subtilis var niger. Three different matrices were used: 3-nitrobenzyl alcohol, picolinic acid and sinapinic acid. A spectrum of gramicidin-S was generated from approximately 250 attomoles of material using a molar ratio of 3-nitrobenzyl alcohol to analyte of approximately 20:1. A single peak, located at 1224 Da, was obtained from the bacterial spores. The washing liquid and extract solution from the spores were analyzed using electrospray mass spectrometry and subsequent MS/MS product ion experiments. This independent analysis suggests that the measured species represents part of the B. subtilis peptidoglycan. The on-line addition of matrix allows quasi-real-time chemical analysis of individual, aerodynamically sized particles, with an overall system residence time of less than 5 seconds. These results suggest that a MALDI-ATOFMS can provide nearly real-time identification of biological aerosols. Copyright 2000 John Wiley & Sons, Ltd.     

The effective temperature of Peptide ions dissociated by sustained off-resonance irradiation collisional activation in fourier transform mass spectrometry

A method for determining the internal energy of biomolecule ions activated by collisions is demonstrated. The dissociation kinetics of protonated leucine enkephalin and doubly protonated bradykinin were measured using sustained off-resonance irradiation (SORI) collisionally activated dissociation (CAD) in a Fourier transform mass spectrometer. Dissociation rate constants are obtained from these kinetic data. In combination with Arrhenius parameters measured with blackbody infrared radiative dissociation, the "effective" temperatures of these ions are obtained. Effects of excitation voltage and frequency and the ion cell pressure were investigated. With typical SORI-CAD experimental conditions, the effective temperatures of these peptide ions range between 200 and 400 degrees C. Higher temperatures can be easily obtained for ions that require more internal energy to dissociate. The effective temperatures of both protonated leucine enkephalin and doubly protonated bradykinin measured with the same experimental conditions are similar. Effective temperatures for protonated leucine enkephalin can also be obtained from the branching ratio of the b(4) and (M + H - H(2)O)(+) pathways. Values obtained from this method are in good agreement with those obtained from the overall dissociation rate constants. Protonated leucine enkephalin is an excellent "thermometer" ion and should be well suited to establishing effective temperatures of ions activated by other dissociation techniques, such as infrared photodissociation, as well as ionization methods, such as matrix assisted laser desorption/ionization.     

Fragmentation of leucine enkephalin as a function of laser fluence in a MALDI TOF-TOF

The effects of laser fluence on ion formation in MALDI were studied using a tandem TOF mass spectrometer with a Nd-YAG laser and alpha-cyano hydrocinnamic acid matrix. Leucine enkephalin ionization and fragmentation were followed as a function of laser fluence ranging from the threshold of ion formation to the maximum available, that is, about 280-930 mJ/mm2. The most notable finding was the appearance of immonium ions at fluence values close to threshold, increasing rapidly and then tapering in intensity with the appearance of typical backbone fragment ions. The data suggest the presence of two distinct environments for ion formation. One is associated with molecular desorption at low values of laser fluence that leads to extensive immonium ion formation. The second becomes dominant at higher fluences, is associated initially with backbone type fragments, but, at the highest values of fluence, progresses to immonium fragments. This second environment is suggestive of ion desorption from large pieces of material ablated from the surface. Arrhenius rate law considerations were used to estimate temperatures associated with the onset of these two processes.     

Laser desorption/ionization techniques in the characterization of high molecular weight oil fractions. Part 1: asphaltenes

The molecular weight distribution of the asphaltene fractions of two types of crude oils from two different Italian fields (samples 1 and 2) was investigated. The analytical tools used to perform these analyses were matrix assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI) mass spectrometry. After observing that the use of the matrix (as well as the addition of Ag+) did not improve the quality of the data compared to that obtained in LDI conditions, all further measurements were performed with the latter technique. Operating under usual conditions of laser power and delay time, a very low resolution was observed, showing only macroscopic differences between the two samples in the molecular weight distribution of the different components. An accurate study on the possible reasons of this undesirable behavior indicates that it can originate from space charge phenomena occurring either in the ion source region or during the flight. A valid parameterization of the delay time and the laser power allowed higher quality spectra to be obtained. Surface-enhanced laser desorption ionization (SELDI) measurements were also performed using normal phase (silica) as the sample holder surface. Under these conditions, better results are obtained, proving that the sample-surface interaction is important to achieve, by means of laser irradiation, a homogeneous set of product ions. Both asphaltene samples were fractionated in five subfractions by gel-permeation chromatography (GPC) to obtain a better separation of the molecular weight distributions; the related spectra confirmed these findings. By using different approaches, relevant and reproducible differences between the asphaltene fractions of the two oil samples were observed.     

Surface induced dissociation of chromium hexacarbonyl on fluorinated alkanethiolate surface in ion cyclotron resonance mass spectrometer: studies of energetics of the process using recursive internal energy distribution search method

Energy transfer in ion-surface collisional activation is characterized for 0–30 eV collisions of chromium hexacarbonyl molecular cations with a monolayer of fluorinated alkanethiolate self-assembled onto a solid gold surface. This surface was mounted on the back trapping plate of the Infinity® cell of a Bruker BioApex 7T ion cyclotron resonance mass spectrometer on the B-field axis orthogonal to the ion beam direction. Internal energy deposition was deduced from fragmentation spectra using a recursive internal energy distribution search method. The efficiency of energy transfer into the ion slowly increases with incident ion energy to a maximum value of 20% at about 23 eV collision energy. Approximate kinetic energy distributions of the fragments were measured by deducing the dependence of ion abundance on trapping potential. From the kinetic energy dependence on mass we infer that rapid decomposition of the molecular cation occurs after it recoils from the surface. Knowledge of both internal and kinetic energy distributions of collisionally activated ions enabled us to deduce the energy deposited into the self-assembled monolayer as a function of collision energy.     

Measurements of mean initial velocities of analyte and matrix ions in infrared matrix-assisted laser desorption ionization mass spectrometry

The mean initial velocities of analyte ions ranging in molecular weight from 1000 Da to 150 kDa and desorbed with a pulsed Er:YAG laser from various solid-state and liquid IR MALDI matrices were measured along with those of the matrix ions. Experiments with UV MALDI were performed for comparison in addition for a 2,5-dihydroxybenzoic acid preparation. Two different measurement principles were employed, (1) a delayed extraction method, relying on the initial velocity-dependent increase of flight times with delay time between laser and HV ion extraction pulse, and (2) a field-free drift method in which the first region of a two-stage ion source was varied in length and the flight times compared. The two methods yielded somewhat different values for the mean initial ion velocities. Based on a detailed discussion of the measurement principles it is suggested that the actual initial velocities of IR MALDI ions lie between the limits set by the two methods. The influences of the analyte-to-matrix ratio, laser fluence, and laser wavelength on the initial ion velocities were also investigated. Significant differences between the desorption mechanisms for liquid and solid-state matrices were observed.     

Mass spectrometric analysis of low molecular mass polyesters by laser desorption/ionization on porous silicon

A low molecular mass polyester was analyzed by desorption/ionization on porous silicon (DIOS) mass spectrometry. The results were compared with those of matrix-assisted laser desorption ionization (MALDI) mass spectrometry using matrixes of alpha-cyano-4-hydroxycinnamic acid (CHCA) and 10,15,20-tetrakis(pentafluorophenyl)porphyrin (F20TPP). The CHCA matrix was not suitable for characterization of low molecular mass components of the polyester because the matrix-related ions interfered with the component ions. On the other hand, the F20TPP matrix showed no interference because no matrix-related ions appeared below m/z 822. However, the solvent selection for determining optimal conditions of sample preparation was limited, because F20TPP does not dissolve readily in any of the available organic solvents. In the DIOS spectra, the polymer ions were observed at high sensitivity without a contaminating ion. No matrix is needed for DIOS spectra of low molecular mass polyesters, facilitating sample preparation and selectivity of a precursor ion in post-source decay measurements.     

Factors affecting the ultraviolet laser desorption of proteins

The production of high-mass quasimolecular ions from proteins by matrix-assisted ultraviolet laser desorption is described. A simple time-of-flight system using a Q-switched frequency-quadrupled Nd-YAG laser to desorb protein molecules is shown to have a mass range of up to 116,000 u by the observation of intact, singly charged quasimolecular ions from 700 fmol of beta-galactosidase subunit (mol.wt = 116,336 Da). Both positive- and negative-ion spectra of proteins are shown. Four new matrix materials, with properties as good as or better than nicotinic acid, are described. A mass resolution of approximately 500 (full width at half maximum definition) is demonstrated for proteins with mol.wt less than 20,000 Da. Product species, formed by fast photochemical reactions in the matrix, are observed to form adduct ions with protein molecules. These adduct ions are a significant cause of the observed broadness of protein quasimolecular ion peaks. The practical physical considerations in detection of large-mass quasimolecular ions from laser desorption, such as detector overloading, are discussed.     

Polarization dependent fragmentation of ions produced by laser desorption from nanopost arrays

Tailored silicon nanopost arrays (NAPA) enable controlled and resonant ion production in laser desorption ionization experiments and have been termed nanophotonic ion sources (Walker et al., J. Phys. Chem. C, 2010, 114, 4835-4840). As the post dimensions are comparable to or smaller than the laser wavelength, near-field effects and localized electromagnetic fields are present in their vicinity. In this contribution, we explore the desorption and ionization mechanism by studying how surface derivatization affects ion yields and fragmentation. We demonstrate that by increasing the laser fluence on derivatized NAPA with less polar surfaces that have decreased interaction energy between the structured silicon substrate and the adsorbate, the spectrum changes from exhibiting primarily molecular ions to showing a growing variety and abundance of fragments. The polarization angle of the laser beam had been shown to dramatically affect the ion yields of adsorbates. For the first time, we report that by rotating the plane of polarization of the desorption laser, the internal energy of the adsorbate can also be modulated resulting in polarization dependent fragmentation. This polarization effect also resulted in selective fragmentation of vitamin B(12). To explore the internal energy of NAPA generated ions, the effect of the post aspect ratios on the laser desorption thresholds and on the internal energy of a preformed ion was studied. Elevated surface temperatures and enhanced near fields in the vicinity of high aspect ratio posts are thought to contribute to desorption and ionization from NAPA. Comparison of the fluence dependence of the internal energies of ions produced from nanoporous silicon and NAPA substrates indicates that surface restructuring or transient melting by the desorption laser is a prerequisite for the former but not for the latter.     

Quadrupole ion trap mass spectrometry of fullerenes

The fullerenes C₆₀ and C₇₀ can be ionized by desorption from a liquid matrix upon bombardment by Cs⁺ ions of 7 keV kinetic energy. The resulting radical cations, when activated in the ion trap by collisions with Xe target, in the presence of helium, undergo extensive dissociation by loss of multiple C₂ units. Large internal energies are deposited into these molecular ions and the dissociation efficiency is in excess of 60%.     

Delayed extraction experiments using a repulsing potential before ion extraction: evidence of non-covalent clusters as ion precursor in UV matrix-assisted laser desorption/ionization. Part II--Dynamic effects with alpha-cyano-4-hydroxycinnamic acid matrix

Delayed extraction experiments were undertaken to gain a better insight into the dynamic effects involved in the ion formation in UV matrix-assisted laser desorption/ionization. Part I1 was devoted to a 2,5-dihydroxybenzoic (2,5-DHB) matrix. The results clearly demonstrated the existence and the role of high-mass precursors corresponding to a non-covalent matrix-analyte association in ion formation. In this complementary study, ion flight time and abundance were studied as a function of the delay extraction time using the matrix alpha-cyano-4-hydroxycinnamic acid (HCCA). Under our instrumental conditions, where ejected ions experienced a low repulsing electric field before extraction, two main results were obtained: (i) two ion components are observed in the peak profiles depending on the repulsing field, a first, major component (I) similar to that observed for 2,5-DHB and a second, minor component (II) apparently triggered by the delayed extraction pulse, and (ii) ion time-of-flight variation vs delay time remained lower than that noted with 2,5-DHB matrix, indicating that the initial axial velocity is smaller. The initial kinetic energy of matrix and low molecular mass peptide ions for the component I is not high enough to overcome the repulsing potential in the delay time range (200-2200 ns) and we have to assume that ions have non-covalent clusters as precursors. Complete desolvation of these clusters-aggregates would be achieved through the extraction step. Simulations of the ion time-of-flight as a function of the delay time allow the determination of the average size of the precursors, typically 4500, 40000 and 50000 u for HCCA, ACTH 7-38 and bovine insulin quasi-molecular ion, respectively, assuming that the precursors are singly charged. The size of these ion precursors is greater than that of those generated for 2,5-DHB. For component II, ions are probably not solvated and they are directly desorbed from the target. Taking into account the results on HCCA and 2,5-DHB matrices and other results from the literature, a general model for ion formation based on clusters as ion precursors is proposed and discussed.     

On-line laser desorption/ionization mass spectrometry of matrix-coated aerosols

Matrix-assisted laser desorption/ionization (MALDI) was used for the on-line analysis of single particles. An aerosol was generated at atmospheric pressure and particles were introduced into a time-of-flight (TOF) mass spectrometer through a single-stage differentially pumped capillary inlet. Prior to entering the mass spectrometer, a matrix was added to the particles using a heated saturator and condenser. A liquid matrix, 3-nitrobenzyl alcohol (NBA), and a solid matrix, picolinic acid (PA), were used. Particles were ablated with a 351 nm excimer laser and the resulting ions were mass-separated in a two-stage reflectron TOF mass spectrometer. Aerosol particles containing the biomolecules erythromycin and gramicidin S were analyzed with and without the matrix addition step. The addition of NBA to the particles resulted in mass spectra that contained an intact molecular ion mass peak. In contrast, PA-coated particles did not yield molecular ion peaks from matrix-coated particles.     

Comparison of the effects of ionization mechanism, analyte concentration, and ion “cool-times” on the internal energies of peptide ions produced by electrospray and atmospheric pressure matrix-assisted laser desorption ionization

The propensities of a series of peptide ions produced by both electrospray and atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) to fragment in an ion trap mass spectrometer under various conditions were studied in detail by measuring the extent of fragmentation of precursor ions by collision induced dissociation (CID) as a function of applied resonance excitation RF voltage. For the most basic peptides, the energy required to fragment MH+ ions generated by electrospray exceeded that required to fragment equivalent AP-MALDI ions under identical instrumental conditions; the reverse was observed for a peptide incorporating no basic residues, while peptides of intermediate basicity showed little difference between the ionization methods. This correlation between peptide basicity and the difference in the energy required to induce fragmentation of MH+ ions generated by AP-MALDI and electrospray is attributed primarily to a trend in the internal energies of the ions generated by AP-MALDI (the greater the difference in gas-phase basicities between the matrix and the analyte the greater the internal energy of the analyte ions produced). Furthermore the internal energies of ions produced by AP-MALDI, but not the equivalent ions formed by electrospray, were observed to decrease with decreasing analyte concentration. We attribute this finding to the cooling effect of endothermic dissociation of analyte ion/matrix molecule clusters following the matrix assisted laser desorption step. Time-resolved analyses (measurement of extent of fragmentation of precursor ions by CID as a function of pre-CID "cool times") revealed that cooling periods in excess of 250 ms were required to achieve internal energy equilibrium through cooling collisions with the helium buffer gas. Furthermore, these analyses demonstrated that, even after these extended cooling times, equivalent ions formed by the two ionization techniques showed different propensities to fragment. We conclude that the two different ionization techniques produce ion populations that may differ in their three-dimensional structure.     

A comparative study of a liquid and a solid matrix in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and collision cross section measurements

We present experimental matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) results comparing a liquid (glycerol/K(4)[Fe(CN)(6)]) and a solid matrix (2,5-dihydroxybenzoic acid, DHB) with respect to analyte signal stability and initial ion velocity. For applications requiring stable production of analyte ions over a long period of time, the liquid matrix is superior to the solid matrix. The stable analyte ion signal obtained from a liquid matrix allowed the measurement of collision cross sections of small poly(ethylene glycol) (PEG(n)) adduct ions in the flight tube with good resolution. The initial velocity of these adduct ions was measured. It was found that analyte molecules from the liquid matrix have initial ion velocities significantly smaller than those from the solid matrix. MALDI-TOF measurements for large molecules using a liquid matrix are therefore likely to result in smaller systematic errors in mass calibrations due to initial ion velocity.     

Matrix-assisted laser desorption/ionization fourier transform ion cyclotron resonance mass spectrometry with pulsed in-source collision gas and in-source ion accumulation

A new matrix-assisted laser desorption/ionization (MALDI) source for Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been developed. The new source is equipped with a hexapole ion guide. The sample on the laser target is one millimeter from the hexapole ion guide, so that ions are desorbed directly into the guide. A device for pulsing collision gas in direct proximity to the laser target makes it possible to cool the ions, which have a kinetic energy spread of several electron volts when produced by the MALDI process. These ions are trapped in the hexapole where positive potentials at the laser target and at an extraction plate help trap ions along the longitudinal axis. After a pre-defined trapping time the voltage of the extraction plate is reversed and the trapped ions are extracted for transmission to the ion cyclotron resonance cell. Accumulation of ions from multiple laser shots in the hexapole before mass spectrometric analysis increases sensitivity. Preliminary sensitivity studies with substance P show that 10 attomoles of analyte applied on the target can be detected with a signal-to-noise (S/N) ratio >15.