Klaus Dreisewerd

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

Abstract 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.

A New Male Sex Pheromone and Novel Cuticular Cues for Chemical Communication in Drosophila

BACKGROUND In many insect species, cuticular hydrocarbons serve as pheromones that can mediate complex social behaviors. In Drosophila melanogaster, several hydrocarbons including the male sex pheromone 11-cis-vaccenyl acetate (cVA) and female-specific 7,11-dienes influence courtship behavior and can function as cues for short-term memory associated with the mating experience. Behavioral and physiological studies suggest that other unidentified chemical communication cues are likely to exist. To more fully characterize the hydrocarbon profile of the D. melanogaster cuticle, we applied direct ultraviolet laser desorption/ionization orthogonal time-of-flight mass spectrometry (UV-LDI-o-TOF MS) and analyzed the surface of intact fruit flies at a spatial resolution of approximately 200 mum. RESULTS We report the chemical and spatial characterization of 28 species of cuticular hydrocarbons, including a new major class of oxygen-containing compounds. Via UV-LDI MS, pheromones previously shown to be expressed exclusively by one sex, e.g., cVA, 7,11-heptacosadiene, and 7,11-nonacosadiene, appear to be found on both male and female flies. In males, cVA colocalizes at the tip of the ejaculatory bulb with a second acetylated hydrocarbon named CH503. We describe the chemical structure of CH503 as 3-O-acetyl-1,3-dihydroxy-octacosa-11,19-diene and demonstrate a behavioral role for this compound as a long-lived inhibitor of male courtship. Like cVA, CH503 is transferred from males to females during mating. Unlike cVA, CH503 remains on the surface of females for at least 10 days. CONCLUSIONS Oxygenated hydrocarbons comprise a major previously undescribed class of compounds on the Drosophila cuticular surface. A newly discovered long-chain acetate, CH503, serves as a mediator of courtship-related chemical communication.

Is the incorporation of analytes into matrix crystals a prerequisite for matrix-assisted laser desorption/ionization mass spectrometry? A study of five positional isomers of dihydroxybenzoic acid

The 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-positional isomers of dihydroxybenzoic (DHB) acid were investigated with respect to their function as matrix-assisted laser desorption/ionization (MALDI) matrices. Optical absorption spectra of solid matrix samples, recorded in diffuse reflection from samples revealed peak broadening and a red shift of the peaks relative to the solution spectra. Single crystals of all isomers were grown from solution and analyzed by x-ray crystallography. Single crystals, as well as standard dried droplet and thin layer preparations with added cytochrome c, were analyzed by UV- (266, 308, 337, 355 nm) and IR- (2.94 μm) MALDI time-of-flight mass spectrometry (MALDI-TOF-MS). A spectrophotometric measurement of the heme absorption around 400 nm of redissolved single crystals showed a quantitative incorporation of the protein into crystals of 2,5-DHB and a partial protein incorporation with large statistical fluctuations into single crystals of 2,4-DHB. No protein incorporation above the detection limit of 10−5 molar analyte-to-matrix ratio was found for the other isomers. Best MALDI spectra from standard preparations were recorded for dried droplet preparations of 2,5-DHB at 337 and 355 nm and for 2,4-DHB at 266 and 308 nm. Both matrices performed well in the IR, too. 2,6-DHB yielded spectra of comparable quality at 337 nm and 2.94 μm, but only when prepared in a thin layer from an acetone solution. The results suggest that protein incorporation into the crystals of solid MALDI matrices is helpful, but not a prerequisite for MALDI. A large surface-to-volume ratio, typical for microcrystalline thin layer preparations supports protein desorption if no measurable incorporation occurs. Undesirable matrix adduct formation to the protein ions was seen for all DHB isomers at the wavelengths of 308 and 266 nm.

Gas-Phase cationization and protonation of neutrals generated by matrix-assisted laser desorption.

The ionization mechanisms involved in matrix-assisted ultraviolet laser desorption/ionization (MALDI) were studied with a time-of-flight mass spectrometer. When protonated or cationized quasimolecular ions generated by MALDI are not extracted promptly, their abundance is a function of the delay time between laser irradiation and ion extraction, maximizing at an optimum delay time (DTM) of a few hundred nanoseconds. The ion abundance at DTM exceeds that of prompt extraction by a factor of 2 or more. Increasing the cation density near the sample surface reduces the DTM, whereas increasing the desorption laser irradiance has the opposite effect. The enhancement suggests extensive gas-phase ion-molecule reactions after irradiation by the desorption laser has ceased.

Pheromonal and Behavioral Cues Trigger Male-to-Female Aggression in Drosophila

By genetically manipulating both pheromonal profiles and behavioral patterns, we find that Drosophila males showed a complete reversal in their patterns of aggression towards other males and females

Mechanisms of energy deposition in infrared matrix-assisted laser desorption/ionization mass spectrometry

Abstract The mechanisms of energy deposition in matrix-assisted laser desorption/ionization mass spectrometry with infrared lasers (IR-MALDI-MS) have been evaluated in a series of experiments. In a first part, the threshold fluences for the generation of IR-MALDI spectra were determined between 2.7 and 4.0 μm wavelength with an optical parametric oscillator as a tunable laser source for nine solid state and two liquid matrices of different chemical structure and compared to the infrared absorption spectra of the compounds. Preliminary spectra of IR-MALDI in the wavelength range of 1.45–1.75 μm are also presented using the overtone vibrations of a glycerol matrix. Matrices were chosen with regard to their IR-MALDI performance and to allow conclusions on the IR-absorption mechanisms. Whereas the wavelength dependence of the threshold fluence for non–hydrogen-bound C–H vibrations essentially follows the absorption spectrum of this functional group, strong discrepancies between the spectral dependence of threshold fluences and IR-absorption spectra were found for the vibrations of O–H and N–H groups around 3-μm wavelength that form strong intermolecular and intramolecular hydrogen bonds. In a second part, experiments are described that interrogate the two most probable mechanisms for the observed deviation of the threshold fluence behavior from the wavelength course of the IR-absorption spectra, that is, absorption by either free or weakened O–H and N–H stretching modes or by residual free water. All investigations were performed with glycerol and succinic acid as examples for common liquid and solid state matrices for IR-MALDI. For glycerol, a fluence-dependent, dynamic change in absorption during the laser pulse was revealed by laser transmission measurements on thin glycerol layers. This effect, characterized by a significant blue shift of the O–H stretch absorption, can be attributed to a weakening of intermolecular hydrogen bonds caused by the transient laser heating of the sample. Taking this effect into account, a good correspondence of the wavelength dependence of the threshold fluence with the infrared absorption under IR-MALDI conditions is derived for glycerol. For succinic acid, in contrast, the identification of the predominant absorption mechanism in the 3-μm wavelength range appears more difficult. A fluence-dependent absorption was not detected in laser transmission experiments with succinic acid single crystals. A change in analyte-to-matrix ratio, with the intention of inducing free absorbers near crystal defects, also did not influence the wavelength dependence of the threshold fluences. However, an influence of the surface-to-volume ratio on the wavelength-dependent threshold fluences was found by a comparison of three different preparation techniques for succinic acid, indicating a putative influence of weakly hydrogen-bound surface absorbers. In combination with the detailed analysis of the wavelength dependence of the threshold fluence given from the first part, a determination of the IR-MALDI process for succinic acid based on the absorption by weakly hydrogen-bound hydroxyl groups is suggested. No evidence for a significant contribution of residual free water absorption to the low-threshold fluences around 3-μm wavelength was found by monitoring a possible change in threshold fluence at the phase transition from water to ice and by reducing the analyte hydration and varying the water content in glycerol preparations. Also, in preparations with frozen hydrated proteins without organic matrices, the wavelength dependence of the threshold fluence did not reflect the spectral absorption of ice, supporting the assumption of a rather minor role of the absorption by residual water in IR-MALDI.

Mass spectrometry imaging with laser-induced postionization

Imaging lipid composition Chemical imaging of cell membranes can be performed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI), but low ionization efficiency often leads to a signal dominated by the main lipid components, such as abundant phosphatidylcholine species. Soltwisch et al. used a tunable laser for post-ionization of neutral species to boost the signal for other membrane components, such as cholesterol and phospho- and glycolipids. Imaging of cells and tissues with these methods allows differentiation based on a more extensive chemical signature. Science, this issue p. 211 The sensitivity of mass spectrometric imaging of membrane lipids is boosted by laser-induced gas-phase ionization. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can simultaneously record the lateral distribution of numerous biomolecules in tissue slices, but its sensitivity is restricted by limited ionization. We used a wavelength-tunable postionization laser to initiate secondary MALDI-like ionization processes in the gas phase. In this way, we could increase the ion yields for numerous lipid classes, liposoluble vitamins, and saccharides, imaged in animal and plant tissue with a 5-micrometer-wide laser spot, by up to two orders of magnitude. Critical parameters for initiation of the secondary ionization processes are pressure of the cooling gas in the ion source, laser wavelength, pulse energy, and delay between the two laser pulses. The technology could enable sensitive MALDI-MS imaging with a lateral resolution in the low micrometer range.

Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer.

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.

Recent methodological advances in MALDI mass spectrometry

AbstractMatrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for characterization of large, thermally labile biomolecules. Advantages of this analytical technique are high sensitivity, robustness, high-throughput capacity, and applicability to a wide range of compound classes. For some years, MALDI-MS has also been increasingly used for mass spectrometric imaging as well as in other areas of clinical research. Recently, several new concepts have been presented that have the potential to further advance the performance characteristics of MALDI. Among these innovations are novel matrices with low proton affinities for particularly efficient protonation of analyte molecules, use of wavelength-tunable lasers to achieve optimum excitation conditions, and use of liquid matrices for improved quantification. Instrumental modifications have also made possible MALDI-MS imaging with cellular resolution as well as an efficient generation of multiply charged MALDI ions by use of heated vacuum interfaces. This article reviews these recent innovations and gives the author’s personal outlook of possible future developments. FigureFigure published in Cramer, RC, Dreisewerd, K. (2007) UV Matrix‐Assisted Laser Desorption/Ionization: Principles, Instrumentation, and Applications. In: M. Gross (Ed.): Encyclopedia of Mass Spectrometry, Vol. 6, pp 646‐661, Elsevier, Oxford

Matrix-assisted laser desorption/ionization with nitrogen lasers of different pulse widths

Abstract The effect of the laser pulse width has been investigated for matrix-assisted laser desorption/ionization (MALDI) of analyte and matrix ions as well as for (photoionized) neutral matrix molecules by comparing two nitrogen lasers (337 nm) with 0.55 and 3 ns pulse widths as commonly used for MALDI mass spectrometry. A homogeneous “flat-top” laser beam profile was used in these experiments. Neither did the threshold fluences (minimum incident laser energy per unit area for producing a spectrum) change, nor was there a difference in the increase in ion signal intensity with laser fluence between the two lasers. Also, no significant differences were found in the mass spectra. The results show that the desorption/ionization process is essentially determined by the energy supplied in the studied pulse width range, rather than by the rate of energy flow into the sample.