Influence of the laser intensity and spot size on the desorption of molecules and ions in matrix-assisted laser desorption/ionization with a uniform beam profile

The dependence of the number of desorbed particles on laser fluence has been investigated for matrix-assisted laser desorption/ionization (MALDI) of analyte and matrix ions as well as for (photoionized) neutral matrix molecules using a homogeneous “flat-top” laser profile. Laser spot diameters ranging from 10 to 200 μm in size have been used. 2,5-Dihydroxybenzoic acid (DHB) and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid) have been tested as matrices. The threshold (for ion detection) is higher and the dependence of the ion signal upon higher-than-threshold fluences is stronger for directly desorbed ions than for photoionized neutral molecules. Directly desorbed analyte ions exhibit the same dependence on fluence as the matrix ions with only minor differences between the two matrices tested, so both have approximately the same detection threshold. For both ions and photoionized neutral molecules, the fluence threshold increases with decreasing spot size while the slope of the intensity/fluence curves decreases. A quasi-thermal, sublimation/desportion model was found to describe the experimental results with excellent precision. For a complete explanation, non-equilibrium effects had to be taken into account.     

Two-photon ionization thresholds of matrix-assisted laser desorption/ionization matrix clusters

Direct two-photon ionization of the matrix has been considered a likely primary ionization mechanism in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This mechanism requires that the vertical ionization threshold of matrix materials be below twice the laser photon energy. Because dimers and larger aggregates may be numerous in the early stages of the MALDI plume expansion, their ionization thresholds are important as well. We have used two-color two-photon ionization to determine the ionization thresholds of jet cooled clusters of an important matrix, 2,5-dihydroxy benzoic acid (DHB), and mixed clusters with the thermal decomposition product of DHB, hydroquinone. The thresholds of the clusters were reduced by only a few tenths of an eV compared to the monomers, to an apparent limit of 7.82 eV for pure DHB clusters. None of the investigated clusters can be directly ionized by two nitrogen laser photons (7.36 eV), and the ionization efficiency at the thresholds is low.     

Titanium dioxide anatase as matrix for matrix-assisted laser desorption/ionization analysis of small molecules

The use of inorganic species as assisting materials in matrix-assisted laser desorption/ionization (MALDI) analysis is an alternative approach to avoid interfering matrix ions in the low-mass region of the mass spectra. Reports of the application of inorganic species as matrices in MALDI analysis of small molecules are, however, scarce. Nevertheless, titanium dioxide (TiO(2)) powder has been reported to be a promising matrix medium. In this study we further explore the use of TiO(2) as a matrix for the MALDI analysis of low molecular weight compounds. We present results showing that nanosized TiO(2) anatase and TiO(2) rutile perform better as MALDI matrices than a commercial TiO(2) anatase/rutile mixture. Moreover, when using nanosized TiO(2) anatase as a matrix, high-quality mass spectra can be obtained with strong analyte signals and weak or non-existing matrix interference ions. Furthermore, our results show that the phase type plays an important role in the application of TiO(2) as a MALDI matrix.     

Miniaturizing sample spots for matrix-assisted laser desorption/ionization mass spectrometry

The trend of miniaturization in bioanalytical chemistry is shifting from technical development to practical application. In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), progress in miniaturizing sample spots has been driven by the needs to increase sensitivity and speed, to interface with other analytical microtechnologies, and to develop miniaturized instrumentation.We review recent developments in miniaturizing sample spots for MALDI-MS. We cover both target modification and microdispensing technologies, and we emphasize the benefits with respect to sensitivity, throughput and automation.We hope that this review will encourage further method development and application of miniaturized sample spots for MALDI-MS, so as to expand applications in analytical chemistry, protein science and molecular biology.     

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.     

Surfactant-mediated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small molecules

A variety of surfactants have been tested as matrix-ion suppressors for the analysis of small molecules by matrix-assisted laser desorption/ionization time-of flight mass spectrometry. Their addition to the common matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) greatly reduces the presence of matrix-related ions when added at the appropriate mole ratio of CHCA/surfactant, while still allowing the analyte signal to be observed. A range of cationic quaternary ammonium surfactants, as well as a neutral and anionic surfactant, was tested for the analysis of phenolics, phenolic acids, peptides and caffeine. It was found that the cationic surfactants, particularly cetyltrimethylammonium bromide (CTAB), were suitable for the analysis of acidic analytes. The anionic surfactant, sodium dodecyl sulfate, showed promise for peptide analysis. For trialanine, the detection limit was observed to be in the 100 femtomole range. The final matrix/surfactant mole ratio was a critical parameter for matrix ion suppression and resulting intensity of analyte signal. It was also found that the mass resolution of analytes was improved by 25-75%. Depth profiling of sample spots, by varying the number of laser shots, revealed that the surfactants tend to migrate toward the top of the droplet during crystallization, and that it is likely that the analyte is also enriched in this surface region. Here, higher analyte/surfactant concentration would reduce matrix-matrix interactions (known to be a source of matrix-derived ions).     

Protein identification with Teflon as matrix-assisted laser desorption/ionization sample support

Protein identification is a critical step in proteomics, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) plays an important role in that identification. Polytetrafluoroethylene (Teflon) was tested as a new MALDI sample support to improve protein identification. The tryptic peptides obtained from a model protein were bound to the surface of a modified MALDI sample holder via the hydrophobic interactions that occur between the Teflon surface and the peptide ion-pairs, and the affinity of alpha-cyano-4-hydroxycinnamic acid for the peptides. During that surface-binding step, the peptide mixture was also desalted and concentrated. A greater number of matched peptides and a larger sequence coverage were obtained for the proteins when Teflon was used as the sample support compared with conventional sample preparation methods and a stainless-steel surface. In addition, the characterization of a small amount of protein was improved with Teflon. Nine silver-stained protein spots obtained from 2-D gel of a human cerebrospinal fluid (CSF) proteome were identified by this method. Among the nine protein spots, peptide 6:c3c fragment and procollagen c-proteinase enhancer were not annotated in any published 2-D map of human CSF. A Teflon MALDI sample support is a low-cost, simple, and effective method that can be used to improve the quality of the MALDI mass spectrum of a complex tryptic peptide mixture, and to achieve a higher level of reliability and success in protein identification.     

Production and fragmentation of multiply charged ions in 'electron-free' matrix-assisted laser desorption/ionization

An unusually large fraction of multiply charged ions is observed in 'electron-free' matrix-assisted laser desorption/ionization (MALDI). Here we investigate how the yield of multiply charged ions depends on experimental parameters in MALDI. It is found to increase if measures are taken to limit the number of electrons in the plume, for example, by using non-metallic MALDI targets or low laser pulse energies. The ionization energy of the matrix is another important parameter that affects the yield of multiply charged ions: matrices with high ionization energies lead to greater intensities of multiply charged ions. It is furthermore proposed that some of the fragment ions observed in MALDI are due to reactions of analyte with electrons in the plume. The possibility of electron capture dissociation of multiply charged ions produced by MALDI is shown.     

Incoherent production reactions of positive and negative ions in matrix-assisted laser desorption/ionization

Utilizing synchronized dual-polarity matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, we found good evidence of the incoherent production of positive and negative matrix ions. Using thin, homogeneous 2,5-dehydroxybenzoic acid (DHB) matrix films, positive and negative matrix ions were found to appear at different threshold laser fluences. The presence of molecular matrix ions of single charge polarity suggests that the existence of DHB ion-pairs may not be a prerequisite in MALDI. Photoelectrons induced by the laser excitation may assist the production of negative DHB ions, as shown in experiments conducted with stainless steel and glass substrates. At high laser fluences, the relative yield of positive and negative matrix ions remained constant when homogeneous matrix films were used, but it fluctuated significantly with inhomogeneous crystal morphology. This result is also inconsistent with the hypothesis that matrix ion-pairs are essential primary ions. Evidence from both low and high laser fluences suggests that the productions of positive and negative matrix ions in MALDI may occur via independent pathways.     

Proton transfer reactions of matrix-assisted laser desorption/ionization matrix monomers and dimers

The gas-phase basicities of monomeric and dimeric deprotonated ferulic and sinapic acids, common matrix-assisted laser desorption/ionization (MALDI) matrices, were determined. A new bracketing method based on structure-reactivity correlations was developed for deriving gas-phase basicities from reaction efficiencies. The matrix dimer anions were found to be significantly less basic than the monomer anions, by about 115 kJ/mol. The low basicity of the dimer anion can qualitatively be explained by resonance stabilization. The energies for proton transfer from dimers to monomers are therefore about 1.2 eV lower than for proton transfer between monomers. For the MALDI process, proton transfer reactions involving matrix dimers provide a low energy pathway for matrix and analyte ion formation.     

Continuous flow infrared matrix-assisted laser desorption/ionization with a solvent matrix

Continuous flow matrix-assisted laser desorption/ionization (MALDI) was demonstrated with infrared laser desorption and an ethanol matrix. A capillary was used to deliver an analyte solution dissolved in ethanol to a metal frit embedded in a sample stage. Typical flow rates were 1.7&mgr;L/min. An optical parametric oscillator tuned to 2.8&mgr;m was used for desorption and ionization, and mass analysis was achieved with a 1 m linear time-of-flight mass spectrometer. Flow injection studies were performed with low picomolar quantities of insulin and myoglobin in solutions containing 0.1 to 1.0% glycerol in ethanol. Copyright 2000 John Wiley & Sons, Ltd.     

Structural effects on polyether cationization by alkali metal ions in matrix-assisted laser desorption/ionization

Structural effects on polyether cationization in matrix-assisted laser desorption/ionization (MALDI) are investigated using three different polyethers: PEG (polyethylene glycol), PPG (polypropylene glycol), and PTHF (polytetrahydrofuran). This study was performed using equimolar cesium and lithium chlorides as the cationizing agent. It was observed that the polyether structure variation led to a substantial change in polyether selectivity for alkali metal ion complexation. Moreover, it was found that like PEG, PPG displays a different selectivity for Cs⁺ and Li⁺ with different matrices. Discussion of these results and their implication in MALDI are given.     

Formulation of matrix solutions for use in matrix-assisted laser desorption/ionization of biomolecules

We report a simple method for converting solid matrices into useful matrix solutions for matrix-assisted laser desorption/ionization (MALDI). This method is based on the dissolution of the solid matrix in a liquid support of low volatility such as glycerol. An appropriate solubilizing reagent was added to promote the dissolution of the matrix materials into the liquid support. Selection of the solubilizing reagent is empirically related to an acid-base relationship, i.e., an acidic solid matrix requires a basic organic compound to form a stable matrix solution in the liquid support and vice versa. A tenfold increase in the solubility can be obtained for many solid matrices when appropriate solubilizing reagents are added into the glycerol support. This solubility enhancement is tentatively attributed to the ion-pair formation in a polar nonvolatile liquid support. In addition, the hydrophobicity of the solid matrix seems to play an important role in the efficiency of the resulting matrix solution. By using glycerol as liquid support, a hydrophilic matrix, such as 2,5-dihydroxybenzoic acid (DHB), showed a substantial “peripheral effect,” in which good analyte ion signals could only be recorded at the peripheral region of the sample droplet. More hydrophobic matrices, such as α-cyano-4-hydroxycinnamic acid (α-CCA), exhibit better and more homogeneous responses at different regions of the droplets. The performance of these matrix solutions was evaluated in terms of the durability, reproducibility, sensitivity, high mass capability, and generality. A typical sample droplet can afford more than an hour of repeated sampling with excellent shot-to-shot reproducibility. A low picomole sensitivity was demonstrated using a luteinizing hormone releasing hormone (LHRH) in a Fourier transform ion cyclotron resonance mass spectrometer with a homemade external MALDI ion source. By using a commercial MALDI time-of-flight mass spectrometer, proteins with masses as high as 66,000 Da were successfully analyzed by using these matrix solutions.     

Thin-layer chromatography--postsource-decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of small drug molecules

The structural analysis of small drug molecules by directly coupling thin-layer chromatography (TLC) with postsource-decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is reported. The applicability of this technique is shown using two examples: the TLC-PSD MALDI analysis of two representatives of nonsteroidal antiinflammatory drugs (tenoxicam and piroxicam) and the analysis of the pharmaceutically active compound UK-137,457 and one of its related substances UK-124,912. The matrices alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) and graphite are used to investigate the effect of the precursor ion selection on the TLC-PSD MALDI spectra of the drug molecules studied. Although alpha-CHCA enhances the [M+H]+ ion formation graphite produces in general only sodium adducts. Structural differentiation of tenoxicam and piroxicam is possible only by selecting the sodium adduct of both drug molecules as precursor ions. In the case of the TLC-PSD MALDI analysis of UK-137,457 and its related substance UK-124,912 at the 1% level, the PSD spectra obtained in alpha-CHCA by selecting the protonated adduct of the small molecules as precursor ions shows distinguishable dissociation patterns containing structurally significant information.     

Internal energies of analyte ions generated from different matrix-assisted laser desorption/ionization matrices

A transfer of energy into the internal modes of the matrix and analyte is expected to occur during matrix-assisted laser desorption/ioniziation (MALDI) processes. Both the physical and thermochemical properties of the MALDI matrix used can influence the ion internal energy and analyte ion fragmentation. Here we report the effect of several MALDI matrices on the relative internal energy of the 2'-deoxyadenylyl-(3',5')-2'-deoxyguanosine (AG) anion. Relative internal energies were probed by low-energy collision-induced dissociation in a Fourier transform ion cyclotron resonance mass spectrometer. Sublimation temperatures of the matrices under study were also determined and found to lie between 409 and 455 K. Analyte ion internal and initial kinetic energies did not correlate with matrix sublimation temperatures. In contrast, a strong correlation between the relative internal energy of the analyte anions and the gas-phase basicity of the matrix anions was found. These results suggest that gas-phase proton transfer reactions play an important role in MALDI analyte ion formation and influence their internal energy and fragmentation behavior. Copyright 2000 John Wiley & Sons, Ltd.