Matrix-assisted laser desorption/ionization tandem reflectron time-of-flight mass spectrometry of fullerenes

Atandem reflectron time-of-flight mass spectrometer developed in our laboratory provides a unique opportunity to investigate the collision-induced dissociation of fullerene ions formed by matrix-assisted laser desorption/ionization (MALDI). Specifically, this opportunity arises from the ability to utilize high energy collisional activation (normally available only on tandem sector instruments by using continuous ionization techniques) for ions formed by pulsed laser desorption, whereas most MALDI time-of-flight instruments record product ion mass spectra of ions formed by metastable or postsource decay. In this study we investigate the products of mass-selected and collisionally activated C ₆₀ ⁺ and C ₇₀ ⁺ ions by using different target gases over a range of target gas pressures. In general, heavier target gases produce more extensive fragmentation and improve the mass resolution of lower mass ionic products because a greater portion of these ions are formed by single collisions. Additionally, the tandem time-of-flight instrument utilizes a nonlinear (curved-field) reflectron in the second mass analyzer that enables high energy collision-induced dissociation spectra to be recorded without scanning or stepping the reflectron voltage.     

Determination of self-exchange rate of alkanethiolates in self-assembled monolayers on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this paper, we describe a new method for determining the exchange rates of alkanethiolates in self-assembled monolayers (SAMs) on gold using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze the compositions of the alkanethiolate in SAMs rapidly and directly. In particular, to investigate the self-exchange of alkanethiols, we prepared a deuterated alkanethiol that has the same molecular properties as the non-deuterated alkanethiol but a different molecular weight. SAMs consisting of deuterated alkanethiolates were immersed in a solution of the non-deuterated alkanethiol, and the influences of the immersion time, temperature, concentration, and solvent on the self-exchange rates were investigated. Furthermore, we assessed the exchange rates among alkanethiols with different carbon chain lengths and different size of ethylene glycol units. In addition, we performed molecular dynamics simulations using a model SAM system in order to understand the molecular mechanism of the exchange process.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of dextran and dextrin derivatives

Application of matrix-assisted laser desorption/ionization (MALDI) to the analysis of dextran and dextrin derivatives, specifically glucose saccharides, by time-of-flight (TOF) mass spectrometry has been reported. MALDI-TOF analysis was carried out on alpha-, beta- and gamma-cyclodextrin, two O-methylated-beta-cyclodextrins of differing degrees of substitution (DS) and dextrans (a linear glucose saccharide), as pure and doped solutions and as mixtures of two or more of these. Doping was carried out with trace amounts of inorganic salts. The purpose of the analysis of the cyclodextrins was to determine whether they would form inclusion complexes with the various cations added, or whether less specific cation addition/exchange was occurring either prior to desorption or in the gas phase.     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of underivatized and permethylated gangliosides

Underivatized and permethylated gangliosides have been studied by the matrix-assisted laser desorption (MALD) ionization technique. The samples investigated included commercially available and highly purified gangliosides from the human brain containing up to five sialic acid residues. Several permethylated gangliosides have also been studied, and MALD has proven successful in analyzing multicomponent mixtures of glycolipids with different fatty acyl residues. During the studies a variety of matrix and wavelength combinations have been tested in both the positive and negative ion modes. The best results have been obtained with the matrices 2,5-dihydroxybenzoic acid, 4-hydrazinobenzoic acid, 1,5-diaminonaphthalene, and 6-aza-2-thiothymine. Negative ion mass spectra of the underivatized gangliosides have always been of better quality than the positive ion mass spectra, exhibiting better signal-to-noise ratio, better resolution, less fragmentation, and less adduct formation with Na⁺ and K⁺. With increasing number of sialic acid substituents the molecular ion region became less and less resolvable, leading to broadened peaks even in the negative ion mode. Fragmentation could frequently be observed in the negative ion mode, and it was pronounced in the positive ion mode. The major fragmentation pathways corresponded to loss of sialyl group(s) and to decarboxylation of one of the sialyl residues. For underivatized gangliosides the typical sample amount used was 10–20 pmol. Permethylation led to a significant improvement in sensitivity (two orders of magnitude); the detection limit of permethylated gangliosides was about 10 fmol. The higher stability of the permethylated compounds was indicated by the fact that positive ion mass spectra exhibited only a marginal extent of fragmentation.     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the analysis of polydienes

An analytical method based on matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry has been developed to provide information on oligomer structure, average molecular weight, and molecular weight distributions of polydienes (e.g., polybutadiene and polyisoprene), an important class of industrial polymers. This MALDI method involves the use of all-trans-retinoic acid as the matrix, copper (II) nitrate as the cationization reagent, and tetrahydrofuran as the solvent. The incorporation of this copper salt generates Cu⁺ adducts with the polymer chains. It also improves the signal strength and extends the upper mass range when used with all-trans-retinoic acid, as compared to silver nitrate. With this formulation, it is demonstrated that polybutadienes of narrow polydispersity with masses up to 300,000 u and polyisoprenes of narrow polydispersity with masses up to 150,000 u can be analyzed. The upper molecular weight limit is set by the requirement of using higher matrix-to-polymer ratios with increasing polymer molecular weight, to the point where the instrument can no longer detect the small quantity of polymer present in the matrix host. It is also shown that this sample preparation generates previously unreported adduction behavior. The practical implications of this adduction behavior on polymer structural analysis, accuracy of molecular weight determination, and the upper molecular weight limit of oligomer resolution are discussed. It is illustrated that, in a linear time-lag focusing MALDI instrument, oligomer resolution can be obtained for polydienes with molecular weights up to 24,000, providing structural confirmation of the end-groups and the repeat unit. The average molecular weights of a number of polydienes of narrow polydispersity determined by MALDI are compared to those obtained by gel permeation chromatography, and discrepancies are noted.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry studies of low molecular weight dendrimers

We report the application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry, with delayed extraction in the reflectron mode, for the characterization of low molecular weight dendrimers. 20 dendrimer samples were measured and 4 typical dendrimers, as examples, are discussed in detail. Several factors that affect the analysis including the matrices used, the concentrations of sample, the solvents and cationization reagent used, were investigated in detail. Experimental results indicate that the type of solvent can greatly influence exact mass measurement. However, sample preparation is generally not very critical for dendrimer analysis using MALDI-ToF since many kinds of matrices and a wide range of sample concentrations can be used efficiently. In addition, the Cs(+) ion can be used to enhance the efficiency of cationization. Some reasons for this behavior are discussed on the basis of results of calculations using Gaussian94 software (a connected system of programs for performing a variety of semi-empirical and ab initio molecular orbital (MO) calculations).     

Matrix-assisted laser desorption/ionization mass spectrometry of taxanes

Taxanes are biologically active compounds that have been extensively used in pharmacology for their powerful anticancer properties. High specificity and low level sensitivity for analysis of these compounds have been obtained with reversed-phase high-pressure liquid chromatography/mass spectrometry (RP-HPLC/MS), but the number of applications of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for low molecular weight analytes is rapidly growing. A new MALDI-MS approach for the rapid screening of a variety of taxanes and a tandem mass spectrometric (MS/MS) analysis of the most important and diagnostic taxane fragmentation pathways are proposed. A solid-phase extraction method followed by preliminary quantification is also reported.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns

Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO(-) from the neutral molecules. Prompt fragment ions [PL-polar head](+) were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for metabolic flux analyses using isotope-labeled ethanol

We describe a novel method for the determination of the concentration and labeling degree of ethanol originating from 1-13C-labeling experiments. This method is suitable for high-throughput metabolic flux analysis because of the possible parallel sample preparation and fast final analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a closed vial containing culture supernatant, ethanol is enzymatically oxidized to acetaldehyde. The acetaldehyde formed evaporates and is readily trapped in a second enclosed but open vial containing acidified 2,4-dinitrophenylhydrazine (DNPH). The 2,4-acetaldehyde dinitrophenylhydrazone (Ac-DNPH) that is formed is insoluble under these conditions. This leads to a constant conversion rate of the acetaldehyde produced from ethanol after 14 h minimum incubation time. MALDI-TOFMS was used to quantify the formed Ac-DNPH with [13C2]-ethanol as internal standard. The relative signal intensities of the unlabeled ethanol derivative as well as of [1-13C]-ethanol were linearly related to the ethanol concentration within a range of 1 to 50 mM with a limit of detection of 0.6 mM, a range which is sufficient for flux analysis in microtiter plate fermentation experiments. The method allows the estimation of the [1-13C]-ethanol originating from 1-13C-labeling experiments of Saccharomyces cerevisiae strains. In experiments where the expected flux range was exceeded, unlabeled ethanol was determined with a linear range from 30 to 500 mM. Ethanol quantification using this method was compared with enzymatic analysis and exhibited differences of less than 3.3% on average. Comparison of flux partitioning ratios between glycolysis and the pentose-phosphate pathway (PPP) based on MALDI-TOFMS and gas chromatography (GC)/MS methods showed good agreement, with differences for ethanol and alanine labeling of only 4.3%.     

Matrix-assisted laser desorption/ionization mass spectrometry using a visible laser

Visible matrix-assisted laser desorption/ionization (VIS-MALDI) was performed using 2-amino-3-nitrophenol as matrix. The matrix is of near-neutral pH, and has an optical absorption band in the near-UV and visible region. A frequency-doubled Nd:YAG laser operated at 532 nm wavelength was used for matrix excitation and comparisons were made with a frequency-tripled Nd:YAG laser (355 nm). Visible and ultraviolet (UV)-MALDI produce similar mass spectra for peptides, polymers, and small proteins with comparable sensitivities. Due to the smaller optical absorption coefficient of the matrix at 532 nm wavelength, the optical penetration depth is larger, and the sample consumption per laser shot in VIS-MALDI is higher than that of UV-MALDI. Nevertheless, VIS-MALDI using 2-amino-3-nitrophenol as matrix may offer a complementary technique to the conventional UV-MALDI method in applications where deeper laser penetration is required.     

Matrix-assisted filament desorption/ionization mass spectrometry

We describe an improved sample preparation method for pulsed filament desorption–ionization mass spectrometry. Samples were deposited in the presence of an excess of liquid or solid matrices. Especially with liquid matrices such as glycerol, this allowed stable and reproducible ion production for a variety of compounds, including biomolecules and synthetic polymers. Substances with molecular weights up to 3000 Da could be desorbed, ionized, and detected by time-of-flight mass spectrometry.     

Matrix-assisted laser desorption/ionization mass spectrometry of collected bioaerosol particles

A method was developed for collection and analysis of bioaerosols by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry using a modified Andersen N6 bioaerosol collector. The overall goal of the study was to develop methods for obtaining mass spectra with minimal reagents and treatment steps for potential use in remote collection and analysis systems. Test bioaerosol particles were generated from a nebulized E. coli bacterial suspension and collected on MALDI targets placed in an Andersen N6 single-stage aerosol impactor. The bioaerosols were mixed with matrix either by deposition on a bare target with the matrix solution added later, or by deposition on a target pre-coated with matrix. The matrix compounds alpha-cyano-4-hydroxycinnamic acid (CHCA) and sinapic acid (SA) were tested and the SA matrix was found to give the best results in number of peaks, resolution, and signal-to-noise ratio. Deposition of bioaerosol particles onto the matrix pre-coated target did not produce signal in the m/z region above 1000, but the signal could be recovered with the addition of a 1:1 (v/v) acetonitrile/water solvent. Addition of solvent by pipette to the pre-coated targets after particle deposition recovered signal comparable to the dried-droplet sample preparations, whereas solvent sprayed into the impactor recovered fewer peaks. Deposition on pre-coated targets with post-collection solvent addition was superior to deposition on bare target followed by post-collection addition of matrix solution.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of some conducting polymers

The application of laser desorption/ionization and matrix-assisted laser desorption/ionization mass spectrometry to the study of the structure of some conducting polymers was investigated. A methodology was developed and the experimental conditions for the characterization of these polymers were established. The parameters chosen to be changed for the method optimization are the matrix material (seven different matrices were tested depending on the polymer studied) and the solvent used in the preparation of the samples. The method seems to be suitable for the study of the structure of two oxidation states of polyaniline: emeraldine and pernigraniline. The analysis of the polymers derived from m-aminophenylbenzothiazole, benzothiazole and tetrathiopentalenes was also performed.} Copyright 2000 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule

This paper introduces a simple, rapid, and sensitive procedure for the analysis of phosphorylated compounds (ROPO(3) (2-)) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method is based on a characteristic mass shift and a total-charge change (from -2 to +1) of the phosphate residue due to complexation of ROPO(3) (2-) with a dinuclear zinc(II) complex (1,3-bis[bis(2-pyridinylmethyl)amino]-2-propanolato dizinc(II) complex, Zn(2)L(3+)) in aqueous solution at physiological pH. Furthermore, the use of single zinc-isotope derivatives ((64)Zn(2)L(3+) and (68)Zn(2)L(3+)) enables improvement of the sensitivity and accuracy of the analysis.     

Small molecule analysis using laser desorption/ionization mass spectrometry on nano-coated silicon with self-assembled monolayers

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an emerging technique for the determination of the molecular weight of biomolecules and their non-covalent complexes without fragmentation. One problem with this technique is the use of excess amounts of matrices, which may produce intense fragment ions and/or clusters at low mass ranges between 1 and 800 Da. These fragments lead to interference, especially concerning the signals of small target molecules. Here, a simple, reusable, and quite inexpensive approach was demonstrated to improve the effectiveness of laser desorption/ionization mass spectrometry (LDI-MS) analysis, especially for small molecules, without using matrix molecules. In this study, substrates with controllable morphologies and thicknesses were developed based on the self-assembly of silane molecules on silicon surfaces using N-(3-trimethoxysilylpropyl)diethylenetriamine (TPDA) and octadecyltrichlorosilane (OTS) molecules. Prepared substrates with nano-overlayers were successfully used in the analysis of different types of small target molecules, namely acrivastine, l-histidine, l-valine, l-phenylalanine, l-arginine, l-methionine and angiotensin I. Our substrates exhibited clear peaks almost without fragmentation for all target molecules, suggesting that these surfaces provide a number of important advantages for LDI-MS analysis, such as ease of preparation, costs, reusability, robustness, easy handling and preventing fragmentation.     

Analysis of transition-metal acetylacetonate complexes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Transition-metal acetylacetonate complexes of the form Metal(acac)(2), where Metal = Fe(II), Co(II), Ni(II), Cu(II), and Zn(II), and Metal(acac)(3), where Metal = V(III), Cr(III), Mn(III), Fe(III), and Co(III), were investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The data was acquired using the aprotic, electron transfer matrix, 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), and the observation of positive radical ions is shown clearly to depend on the metal element and the oxidation state it occupies. The ionization energy of DCTB was calculated to be 8.08 eV by density functional theory methods, which is notably lower than the experimental value, but within the range of other computational values. This value is very close to those of the analytes, so the existing electron transfer mechanism which is based on the ionization energies of the matrix and analyte, cannot be used predictively. Similarly, the data neither proves nor disproves the validity of the existing electron transfer ionization mechanism, with respect to metal coordination complexes without strong chromophores. In this case, periodic trends may be more useful in explaining the observed species and the prediction of species from sets of similar complexes. The addition of a sodium salt benefits the MALDI-TOFMS characterization of certain compounds studied, but the benefit of the addition of ammonium or silver salts is negligible.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an inorganic particle matrix for small molecule analysis

Fine metal or metal oxide powder as an alternative to conventional organic matrices in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been utilized successfully for lower molecular mass analytes, poly(ethylene glycol) 200 (PEG 200) and methyl stearate. Eleven kinds of particle, Al, Mn, Mo, Si, Sn, SnO2, TiO2, W, WO3, Zn and ZnO, were evaluated. The analyte was mixed with a metal or metal oxide powder (inorganic matrix) with particle diameter of tens of micrometers and liquid dispersant, followed by application to the sample target. Using a commercial MALDI-TOFMS instrument equipped with an internal 337 nm pulsed nitrogen laser, the analytes, PEG 200 and methyl stearate, were ionized as the alkali metal ion adducted molecules [M+Na]+ or [M+K]+ when the inorganic matrices Mn, Mo, Si, Sn, TiO2, W, WO3, Zn or ZnO were used. In the case of an Al matrix, PEG 200 was ionized as [M+K]+, whereas methyl stearate was ionized as [M+H]+ and [M+Al]+. These particles have potential as the matrix for MALDI. During our examination, however, only SnO2 particles did not ionize either PEG 200 or methyl stearate. Based on our protocol, when TiO2 powder was suspended with liquid paraffin, PEG 200 and methyl stearate gave their MALDI-TOF mass spectra with the lowest background noise and highest intensity. TiO2 powder seemed to be a broad potential matrix for low molecular mass polar or non-polar analytes. The results suggested that bulk particles caused rapid heating/vaporization processes and ionized analyte molecules under irradiation with a pulsed UV laser. The present method can be readily applied to obtain the low background noise MALDI-TOF mass spectra of small-sized compounds.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry monitoring of anthocyanins in extracts from Arabidopsis thaliana leaves

Anthocyanins are secondary plant metabolites ubiquitous in the plant kingdom. They have different biological activities, so monitoring their content in plant tissue or in feed/food derived from plants may be an important task in different projects from various fields of molecular biology and biotechnology. Profiling of secondary metabolites with high-performance liquid chromatography/mass spectrometry (HPLC/MS) systems is time-consuming, especially when many samples have to be checked within a defined time frame with a reasonable number of repetitions according to the metabolomic standards. Even application of the advanced ultra-performance liquid chromatography (UPLC)/MS or equivalent systems would require a long time for analysis of numerous samples. We demonstrate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the assessment of level (concentration) of anthocyanins in leaf tissues of four Arabidopsis thaliana ecotypes grown at normal (20 degrees C/16 degrees C day/night) and decreased (4 degrees C) temperature. The quantitative results were obtained for anthocyanins with MALDI-TOF MS using ferulic acid as a matrix. The amounts of anthocyanins in leaves of A. thaliana varied from 0.3-2.5 microg per gram of leaves for ecotypes Col-0 and C24, respectively, and contents of these markedly increased in plants grown in the cold. The applied analytical method exhibited better repeatability of measurements than obtained with an HPLC/ion trap MS system.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer analysis: solvent effect in sample preparation

The success of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for the characterization of polymer structures and for the determination of average molecular weights and distributions depends on the use of a proper sample/matrix preparation protocol. This work examines the effect of solvents, particularly solvent mixtures, used to prepare polymer, matrix, and cationization reagent solutions, on MALDI analysis. It is shown that the use of solvent mixtures consisting of polymer solvents does not have a significant effect on the molecular weight determination of polystyrene 7000 and poly(methyl methacrylate) 3750. However, solvent mixtures containing a polymer nonsolvent can affect the signal reproducibility and cause errors in average weight measurement. This solvent effect was further investigated by using confocal laser fluorescence microscopy in conjunction with the use of a fluorescein-labeled polystyrene. It is demonstrated that sample morphology and polymer distribution on the probe can be greatly influenced by the type of solvents used. For sample preparation in MALDI analysis of polymers, it is important to select a solvent system that will allow matrix crystallization to take place prior to polymer precipitation. The use of an excess amount of any polymer nonsolvent should be avoided.