Ultraviolet Matrix-assisted Laser Desorption Research Articles

Infrared MALDI Mass Spectrometry with Laser-Induced Postionization for Imaging of Bacterial Colonies.

Ultraviolet matrix-assisted laser desorption ionization mass spectrometry imaging (UV-MALDI-MSI) is a powerful tool to visualize bacterial metabolites in microbial colonies and in biofilms. However, a challenge for the method is the efficient extraction of analytes from deeper within the bacterial colonies and from the cytoplasm of individual cells during the matrix coating step. Here, we used a pulsed infrared (IR) laser of 2.94 μm wavelength to disrupt and ablate bacterial cells without a prior coating with a MALDI matrix. Instead, tissue water or, in some experiments, in addition a small amount of glycerol was exploited for the deposition of the IR laser energy and for supporting the ionization of the analytes. Compared to water, glycerol exhibits a lower vapor pressure, which prolonged the available measurement time window within an MSI experiment. Mass spectra were acquired with a hybrid Synapt G2-S HDMS instrument at a pixel size of 120 μm. A frequency-quadrupled q-switched Nd:YAG laser with 266 nm wavelength served for laser-induced postionization (MALDI-2). In this way, the ion abundances of numerous small molecules such as nucleobases, 2-alkyl-quinolones, a prominent class of Pseudomonas aeruginosa signaling molecules involved in one of the three quorum-sensing pathways, and also the signals of various bacterial phospholipids were boosted, partially by orders of magnitude. We analyzed single and cocultured colonies of Gram-negative P. aeruginosa and of Gram-positive Bacillus subtilis and Staphylococcus aureus as exemplary bacterial systems. To enable a rapid (within 5 s) MSI-compatible steam inactivation in a custom-made autoclave filled with hot water steam, bacterial cultures were grown on porous polyamide membranes. Compared to a UV-MALDI-2-MS measurement of the same systems, mass spectra with a reduced low mass background were generally generated. This resulted in the unequivocal detection of numerous metabolites only with the IR laser. In a fundamental part of our study, and to optimize the IR-MALDI-2 approach for the highest analytical sensitivity, we characterized the expansion dynamics of the particle plume as generated by the IR laser. Here, we recorded the total ion count and the intensities of selected signals registered from P. aeruginosa samples as a function of the interlaser delay and buffer gas pressure in the ion source. The data revealed that the IR-MALDI-2 ion signals are primarily generated from slow particles having mean velocities of ∼10 m/s. Interestingly, two different pressure/delay time regimes of the optimized ionization efficiency for phospholipids and smaller metabolites, respectively, were revealed, a result pointing to yet-unknown convoluted reaction cascades. The described IR-MALDI-2 method could be a helpful new tool for a microbial mass spectrometry imaging of small molecules requiring little sample preparation.

MALDI- and LDI-MS saponin fingerprint of leaves and stick components of commercial yerba mate (Ilex paraguariensis).

Yerba mate (YM) is massively produced and consumed as an infusion in South America and spreading all over the world. This product is obtained from dried leaves of Ilex paraguariensis Saint Hilaire, mixed with fragments of dried branches (sticks). For its commercialization, YM must have a minimum percentage of leaves because its presence determines YM quality and price. Till today, a mechanical methodology to determine the relative amount of components (sticks, leaves, and powder) is used. There is not any modern analytical method that provides information for quick characterization of the YM components. Typical saponin fingerprints for leaves and sticks were found by using ultraviolet matrix-assisted laser desorption ionization and ultraviolet laser desorption ionization mass spectrometry. Then, their possible application as useful tools for quick characterization of components of commercial YM (leaves and sticks) is presented. Furthermore, fingerprints obtained from authentic samples of Ilex paraguariensis and Ilex dumosa are also included and discussed. Each Ilex show typical saponin fingerprints for leaves and sticks.

Biospectroscopy, biospectrometry and imaging of Ilex paraguariensis. Basis for non-destructive quality evaluation using artificial vision.

Yerba mate (YM) is massively produced and consumed as an infusion in South America and is gaining popularity all over the world. This product is obtained from the dried leaves of Ilex paraguariensis Saint Hilaire, mixed with fragments of its dried branches. For its commercialization YM must have a minimum percentage of leaves according to a standard classification. Until now, composition quantification has been mechanically performed, thus development of new methods is still pending. In this work a quantification method using solid-phase molecular fluorescence, alternately diffuse reflectance spectroscopy and an imaging technique using a scanner have been proposed. Strong differences between the spectroscopic properties of leaves and sticks were observed and in all the cases linear correlations between the processed signals and composition were observed. Interesting differences in chemical composition of YM leaves and sticks were additionally obtained in this work by means of total phenol content quantification, ultraviolet matrix assisted laser-desorption ionization mass spectrometry and ultraviolet laser-desorption ionization mass spectrometry.

Thermal Proton Transfer Reactions in Ultraviolet Matrix-Assisted Laser Desorption/Ionization

One of the reasons that thermally induced reactions are not considered a crucial mechanism in ultraviolet matrix-assisted laser desorption ionization (UV-MALDI) is the low ion-to-neutral ratios. Large ion-to-neutral ratios (10(-4)) have been used to justify the unimportance of thermally induced reactions in UV-MALDI. Recent experimental measurements have shown that the upper limit of the total ion-to-neutral ratio is approximately 10(-7) at a high laser fluence and less than 10(-7) at a low laser fluence. Therefore, reexamining the possible contributions of thermally induced reactions in MALDI may be worthwhile. In this study, the concept of polar fluid was employed to explain the generation of primary ions in MALDI. A simple model, namely thermal proton transfer, was used to estimate the ion-to-neutral ratios in MALDI. We demonstrated that the theoretical calculations of ion-to-neutral ratios exhibit the same trend and similar orders of magnitude compared with those of experimental measurements. Although thermal proton transfer may not generate all of the ions observed in MALDI, the calculations demonstrated that thermally induced reactions play a crucial role in UV-MALDI.

Matching the laser wavelength to the absorption properties of matrices increases the ion yield in UV-MALDI mass spectrometry

A high analytical sensitivity in ultraviolet matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is only achieved if the laser wavelength corresponds to a high optical absorption of the matrix. Laser fluence and the physicochemical properties of the compounds, e.g., the proton affinity, also influence analytical sensitivity significantly. In combination, these parameters determine the amount of material ejected per laser pulse and the ion yield, i.e., the fraction of ionized biomolecules. Here, we recorded peptide ion signal intensities as a function of these parameters. Three cinnamic acid matrices were investigated: α-cyano-4-hydroxycinnamic acid, α-cyano-4-chlorocinnamic acid, and α-cyano-2,4-difluorocinnamic acid. In addition, 2,5-dihydroxybenzoic acid was used in comparison experiments. Ion signal intensities "per laser shot" and integrated ion signal intensities were acquired over 900 consecutive laser pulses applied on distinct positions on the dried-droplet sample preparations. With respect to laser wavelength, the two standard MALDI wavelengths of 337/355 nm were investigated. Also, 305 or 320 nm was selected to account for the blue-shifted absorption profiles of the halogenated derivatives. Maximal peptide ion intensities were obtained if the laser wavelength fell within the peak of the absorption profile of the compound and for fluences two to three times the corresponding ion detection threshold. The results indicate ways for improving the analytical sensitivity in MALDI-MS, and in particular for MALDI-MS imaging applications where a limited amount of material is available per irradiated pixel.

Selective Screening of Tyrosine-Nitrated Peptides in Tryptic Mixtures by In-Source Photodissociation at 355 nm in Matrix-Assisted Laser Desorption Ionization

Nitration of tyrosine residues in proteins is an important post-translational modification related to various diseases such as Alzheimer's. In this work, efficient and selective photodissociation (PD) at 355 nm was observed for [M + H](+), [M + H - 16](+), and [M + H - 32](+) generated by matrix-assisted ultraviolet laser desorption ionization (UV-MALDI) of tyrosine-nitrated peptides (nitropeptides). Product ion spectra obtained by post-source PD at this wavelength contained useful information on the amino acid sequence. The spectra for nitropeptides obtained with 355 nm irradiation inside the ion source (MALDI/in-source PD) displayed characteristic triplet patterns due to PD of the above ions. For peptides displaying prominent signal in a MALDI mass map of a tryptic mixture, which are mostly those with arginine at the C-terminus, in-source PD allowed positive identification of their tyrosine-nitrated forms. Identification of such nitropeptides was possible at the 10 fmol level (in tryptic digest of 100 fmol BSA).

Negative ion ultraviolet matrix-assisted laser desorption ionization mass spectrometry and post source decay of glycosyl esters of nucleoside pyrophosphates.

Six different glycosyl esters of nucleoside pyrophosphates (monosaccharide nucleotides) were analyzed by means of matrix-assisted laser desorption/ionisation reflectron time-of-flight mass spectrometry (MALDI/RToF MS) in the negative ion mode. Several matrices were evaluated and 3-hydroxypicolinic acid as well as alpha-cyano-4- hydroxycinnamic acid (CHCA) turned out to be the matrices of choice applying the thin layer technique to obtain maximum sensitivity for deprotonated molecular ion detection and maximal fragmentation particular with CHCA. The determination of the molecular mass with a mass accuracy below 0.1% was feasible with sample amounts in the lower femtomole range applying a MALDI desk-top mass spectrometer. A further important refinement of this technique was the use of post source decay (PSD) fragment ion analysis with a curved field reflector (which means no stepping of the reflector voltage). Detailed structural information of the six selected monosaccharide nucleotides could be obtained with PSD and differences in the fragmentation pattern were used to distinguish them. This method (based on molecular mass and PSD fragment ion analysis) has been applied to verify the presence of a glycosyl ester of nucleoside pyrophosphate in samples from Saccharomyces cerevisiae.

Cyanogen Bromide Peptides of the Fibrillar Collagens I, III, and V and Their Mass Spectrometric Characterization: Detection of Linear Peptides, Peptide Glycosylation, and Cross-Linking Peptides Involved in Formation of Homo- and Heterotypic Fibrils

The network of the fibrillar collagens I, III, and V, extracted from fetal calf skin and cleaved with cyanogen bromide, was studied by means of ultraviolet matrix-assisted laser desorption ionization time-of-flight mass spectrometry (UV-MALDI MS). Nearly all of the expected cyanogen bromide peptides of the different alpha chains were detected. Distinct peptides are identified that can serve as a reference signal for the individual alpha-chains. Homo- and heterotypic cross-linking patterns, some of which have not been described before for bovine collagen, are indicated by comparison of the mass spectrometric data with documented amino acid sequences. Potential cross-linking mechanisms are discussed. For example, the mass spectrometric data suggest that the formation of heterotypic I/III and I/V fibrils is substantially determined by the telo-regions of type I collagen, which are covalently connected to the corresponding helical and nonhelical cross-linking domains of adjacent molecules either by 4D or 0D-stagger bonds. The chemical nature of the cross-links can be concluded. The data also indicate a disturbed formation of heterotypic fibrils. Finally, collagen glycosylation can also be identified.

Effects of silsesquioxane coating structure on mechanical interfacial properties of carbon fibre/polyarylacetylene composites

Carbon fibres (CF) were treated with different coatings, including [3-(methacryloxy)propyl]trimethoxylsilane (MPMS), [3-(methacryloxy)propyl]silsesquioxane (MPMS-SSO), and (methacryloxy)propyl polyhedral oligomeric silsesquioxane (Methacryl-POSS), to improve the interfacial properties of carbon fibre reinforced polyarylacetylene (PAA) matrix composites. MPMS-SSO was obtained from the hydrolytic condensation of MPMS. The complicated structure, including cage and ladder one, of MPMS-SSO may be assigned by Fourier transforms infrared (FT-IR) spectra, 1H, 13C and 29Si nuclear magnetic resonance (NMR) and ultraviolet matrix-assisted laser desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS). Interlaminar shear strength (ILSS) was tested to investigate the effect of coating structure on the interfacial bonding. The values of ILSS of untreated and treated CF/PAA composites with different coatings (MPMS, MPMS-SSO and Methacryl-POSS) show that the treatment effect of Methacryl-POSS coating is the best one and the MPMS-SSO coating is better than that of MPMS coating. SEM micrographs of shear fracture of CF/PAA composites also suggested the different coating treatment effects. The differences of increasing degrees of ILSS indicate that the structure of coating is important when silsesquioxanes are used as coatings to treat fibre surface for building up the adhesion and improving interfacial properties of fibre reinforced polymer matrix composites.

Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro and in vivo

BackgroundAgaro-oligosaccharides derived from red seaweed polysaccharide have been reported to possess antioxidant activity. In order to assess the live protective effects of agar-oligosaccharides, we did both in vitro and in vivo studies based on own-made agaro-oligosaccharides, and the structural information of this oligosaccharide was also determined.MethodStructure of agaro-oligosaccharides prepared with acid hydrolysis on agar was confirmed by matrix-assisted ultraviolet laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and NMR. The antioxidant effect of agaro-oligosaccharides on intracellular reactive oxygen species (ROS) was assessed by 2', 7'-dichlorofluorescin diacetate. Carbon tetrachloride was used to induce liver injury, some index including SOD, GSH-Px, MDA, AST, ALT were examined to determine the hepatoprotective effect of agaro-oligosaccharides.ResultsAgaro-oligosaccharides we got were composed of odd polymerizations with molecular weights ranged from 500 to 2500. Results from intracellular test indicated that agaro-oligosaccharides could significantly scavenge the level of oxidants in the hepatocytes, more beneficially, also associated with the improvement of cell viability In vivo studies of the antioxidant effects on tissue peroxidative damage induced by carbon tetrachloride in rat model indicated that agaro-oligosaccharides could elevate the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and decrease the level of malondialdehyde (MDA), glutamate oxaloacetate transaminase (AST), glutamic pyruvic transaminase (ALT) significantly. At 400 mg/kg, MDA level reduced 44 % and 21 % in liver and heart, SOD and GSH-Px increased to highest in liver and serum, while ALT level decreased 22.16 % in serum.ConclusionOverall, the results of the present study indicate that agaro-oligosaccharides can exert their in vitro and in vivo hepatoprotective effect through scavenging oxidative damage induced by ROS.

Lipid imaging by gold cluster time-of-flight secondary ion mass spectrometry: application to Duchenne muscular dystrophy

Imaging with time-of-flight secondary ion mass spectrometry (TOF-SIMS) has expanded very rapidly with the development of gold cluster ion sources (Au(3+)). It is now possible to acquire ion density maps (ion images) on a tissue section without any treatment and with a lateral resolution of few micrometers. In this article, we have taken advantage of this technique to study the degeneration/regeneration process in muscles of a Duchenne muscular dystrophy model mouse. Specific distribution of different lipid classes (fatty acids, triglycerides, phospholipids, tocopherol, coenzyme Q9, and cholesterol) allows us to distinguish three different regions on a mouse leg section: one is destroyed, another is degenerating (oxidative stress and deregulation of the phosphoinositol cycle), and the last one is stable. TOF-SIMS imaging shows the ability to localize directly on a tissue section a great number of lipid compounds that reflect the state of the cellular metabolism.

Matrix-assisted ultraviolet laser-desorption ionization and electrospray-ionization time-of-flight mass spectrometry of sulfated neocarrabiose oligosaccharides

Several commercial sulfated neocarrabiose oligosaccharides were analyzed by matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS). UV-MALDI-TOF-MS was carried out in the linear and reflectron modes and, as routine, in both the positive- and negative-ion modes. 2,5-Dihydroxybenzoic acid and nor-harmane were used as matrices. In the positive- and negative-ion modes, with both matrices, peaks corresponding to (M+Na) + and (M−Na) − ions, respectively, were obtained, with only some signals due to glycosidic linkage cleavages (prompt fragmentation). With 2,5-dihydroxybenzoic acid abundant matrix signals were observed; nor-harmane afforded very few matrix signals in both ion modes, but more desulfation (prompt fragmentation) of the compounds occurred. When the desorption/ionization process was highly efficient, the post-source decay (PSD) fragmentation patterns were also investigated; most of the fragments detected derived from glycosidic linkage cleavages. Electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) in the negative-ion mode confirmed, with the observation of the (M−Na) − and the multiply charged anions, the identity and the purity of the samples.

Modeling the cluster formation during infrared and ultraviolet matrix-assisted laser desorption ionization of oligonucleotides in succinic acid matrix with molecular mechanics

A large succinic acid (HOOC(CH2)2COOH) matrix containing 7 × 7 × 7 unit cells with guest oligonucleotide AGCAGCT was modeled with molecular dynamics simulation for infrared matrix-assisted laser desorption ionization. We simulated the laser heating of the succinic acid (this data is still missing from the literature) with λ=2.94 μm infrared laser pulses and compared it to ultraviolet excitation. We did this in order to elucidate the cluster formation of succinic acid in the gas phase in itself and around the analyte. At this wavelength, the laser energy is coupled into the matrix through the OH vibrations of the carboxyl groups. The most pronounced difference we observed at 1,500 K simulation is that infrared heating generates about 10–15 more succinic acid molecules bound to the analyte in noncovalent complex form than the ultraviolet mode, which generates about 2 molecules thus bound. We report energy redistribution within the matrix between the host and guest species as well as other dynamical properties. The parameter and topology data for succinic acid that we used are reported and ready for use in CHARMM computer code environment for simulation.

Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry of sulfated mannans from the red seaweed Nothogenia fastigiata

Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) was applied to sulfated xylo-mannan fractions from Nothogenia fastigiata in order to determine their molecular weights and distribution profiles. The number-average molecular weight calculated from the spectra was similar to that determined by chemical end-group analysis for the lower molecular weight fractions. For the other fractions, the number-average molecular weight was lower than that chemically determined; the increased difference may be attributed to higher desorption difficulties and, consequently, mass-dependent discrimination. A reconstructed spectrum, using the peaks obtained from all the fractions, suggested an unimodal distribution. The best results were obtained by using 2,5-dihydroxybenzoic acid as matrix doped with 1-hydroxyisoquinoline and with harmane and nor-harmane.

Diffusive transfer to membranes as an effective interface between gel electrophoresis and mass spectrometry

Diffusive transfer was examined as a blotting method to transfer proteins from polyacrylamide gels to membranes for ultraviolet matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The method is well-suited for transfers from isoelectric focusing (IEF) gels. Spectra have been obtained for 11 pmol of 66 kDa albumin loaded onto an IEF gel and subsequently blotted to polyethylene. Similarly, masses of intact carbonic anhydrase and hemoglobin were obtained from 14 and 20 pmol loadings. This methodology is also compatible with blotting high molecular weight proteins, as seen for 6 pmol of the 150 kDa monoclonal antibody anti-β-galactosidase transferred to Goretex. Polypropylene, Teflon, Nafion and polyvinylidene difluoride (PVDF) also produced good spectra following diffusive transfer. Only analysis from PVDF required that the membrane be kept wet prior to application of matrix. Considerations in mass accuracy for analysis from large-area membranes with continuous extraction and delayed extraction were explored, as were remedies for surface charging. Vapor phase CNBr cleavage was applied to membrane-bound samples for peptide mapping.

Positive ion formation in the ultraviolet matrix-assisted laser desorption / ionization analysis of oligonucleotides by using 2,5-dihydroxybenzoic acid

The development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and its demonstrated performance with large proteins has generated substantial interest in utilizing this technique as an alternative to gel electrophoresis for DNA sequence analysis. However, a lack of understanding of the desorption and ionization processes has greatly hampered advances in this field. This article explores the formation of positively charged oligonucleotides in UV (355-nm) MALDI analysis by using the matrix 2,5-dihydroxybenzoic acid. Whereas substantial fragmentation is observed in the positive-ion mode by using the short oligomer d(TAGGT), no fragmentation is evident in the negative-ion mode under identical conditions. The fragmentation products are consistent with a previously published model in which base protonation initiates base loss, which leads to subsequent cleavage of the phosphodiester backbone. Several polydeoxythymidilic acids containing modified nucleosides were used to investigate positive-ion formation. The results support the hypothesis that positive ions are formed by protonation of the nucleobases. Because base protonation initiates base loss, fragmentation is intrinsic to positive-ion formation in the MALDI analysis of oligonucleotides. This result explains the dramatic difference in fragmentation observed in positive-ion compared to negative-ion UV-MALDI mass spectra of oligonucleotides.

β-Carboline Alkaloids as Matrices for Matrix-assisted Ultraviolet Laser Desorption Time-of-flight Mass Spectrometry of Proteins and Sulfated Oligosaccharides: a Comparative Study Using Phenylcarbonyl Compounds, Carbazoles and Classical Matrices

β-Carboline Alkaloids as Matrices for Matrix-assisted Ultraviolet Laser Desorption Time-of-flight Mass Spectrometry of Proteins and Sulfated Oligosaccharides: a Comparative Study Using Phenylcarbonyl Compounds, Carbazoles and Classical Matrices

The analysis of myocardial proteins by infrared and ultraviolet laser desorption mass spectrometry.

The use of infrared (IR) and ultraviolet (UV) matrix-assisted laser desorption (MALDI) mass spectrometry to analyse myocardial proteins separated by two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) is discussed. Proteins were electroblotted onto a FluoroTrans polyvinylidene difluoride (PVDF) membrane in order to facilitate analysis by MALDI, which represented the most efficient means of extracting large numbers of proteins simultaneously. Once on a FluoroTrans membrane, IR-MALDI was used to obtain spectra from selected protein spots, but no useful signals were obtained with UV MALDI. Spectra were generated from 46 of 50 spots analysed with protein masses from 13 to 82 kDa and isoelectric points (pI) 4.7-7.8. For those protein spots that had previously been characterised, and for which both sequence and post-translational modification data were known, IR-MALDI data was within plus or minus 0.5% of the expected mass. Some spots contained more than one protein signal, illustrating the increased information obtainable from MALDI, but also suggesting the limit of resolution of 2-D gels for separating large numbers of proteins. Attempts to digest proteins with specific proteases and generate peptide mass fingerprints by MALDI analysis on the membrane were unsuccessful with either IR or UV lasers. The peptides were extracted from the membrane and readily analysed by UV MALDI for peptide spectra. Poor data was obtained for peptide digests with IR-MALDI, probably because of matrix suppression by digest buffer. In order to obtain the maximum amount of information from blotted proteins, both IR and UV MALDI were required.

Analysis of hydrocarbon dendrimers by laser desorption time-of-flight and Fourier transform mass spectrometry

The first mass spectrometric analysis of a new class of hydrocarbon dendrimers that result from a convergent synthetic approach is reported. Molecular weights of a series of phenylacetylene dendrimers (715 to 14776 u MW) are characterized by ultraviolet matrix-assisted laser desorption (MALD) time-of-flight (TOF) mass spectrometry, direct and silver chemical ionization infrared laser desorption Fourier transform mass spectrometry (FTMS), and ultraviolet matrix-assisted laser desorption silver chemical ionization Fourier transform mass spectrometry. New matrices and techniques were developed to facilitate analysis of the dendrimers. Mass measurement accuracies between 10 and 25 ppm are obtained for molecular ion species of the five dendrimers analyzed. Laser desorption time-of-flight and FTMS techniques are shown to be complementary, with FTMS providing high mass resolution (27,000–67,000 resolving power) and accuracy for lower mass dendrimers (10–14 ppm) and MALD TOF yielding the highest resolution (1100 resolving power) and accuracy (25 ppm) for the largest dendrimer. These results are consistent with proposed empirical formulas.

Initial velocities of positive and negative protein molecule-ions produced in matrix-assisted ultraviolet laser desorption using a liquid matrix

The initial axial velocities for the positive and negative cytochrome c molecule-ions desorbed from a liquid matrix (3-nitro-benzyl alcohol) using a 17 ns ultraviolet laser (266 nm) have been determined. The method employed was based upon measurement of ion flight times through a field-free path co-axial with the ion optical axis. The possible interferences, such as energy deficits, due to the presence of electric fields were avoided by using a grid-electrode placed at a very short distance from the sample stage. The potential of this electrode was maintained at the same potential as the sample stage, thereby creating a narrow field-free region within which ion generation took place. Systematic variations of the potential at a second grid-electrode caused a gradual shift of the ion flight times, which could be related to the initial ion velocities. The ion velocities were obtained by correlating the measured time-shifts with values from theoretical analysis. The positive and negative molecule-ions were found to have axial velocities of 840±70 and 750±40 ms −1, respectively.