Investigation of UV matrix-assisted laser desorption fourier transform mass spectrometry for peptides.

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Ultraviolet matrix-assisted laser desorption can be used to enhance formation of [M + H](+), [M + Na](+), and [M + K)(+) ions from small peptides for Fourier transform mass spectrometry (FTMS). In accord with laser desorption (LD) time-of-flight experiments, matrices such as nicotinic acid and 2-pyrazinecarboxylic acid exhibit strong enhancement effects (i.e., formation of abundant protonated and cationized molecules for the analyte with virtually no fragment ions) for 266 nm LD/FTMS, whereas pyrazinedicarboxylic acid provides no matrix enhancement at this wavelength. Both sinapinic acid and coumarin-120 provide strong matrix enhancement effects for the 355-nm LD of peptides. For the small peptides examined in this study, no significant differences in the abundance of fragment ions were observed between the 266- and 355-nm wavelengths. Matrix-assisted LD/FTMS is useful for the generation and characterization of ions corresponding to protonated and cationized molecules from virtually all biological compounds with molecular weights up to 2000. The lack of observation of biological ions with m/ z > 2500 may be related to inefficient trapping of these laser-desorbed ions or instrumental detection limitations of FTMS and is under further investigation.

Methyl guanine isomer distinction by hydrogen / deuterium exchange using a fourier transform mass spectrometer.

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Differentiation of the seven isomers of methyl guanine has been accomplished by monitoring gas-phase hydrogen/deuterium (H/D) exchange reactions of the protonated molecular ions with deuterium oxide (D2O) in a Fourier transform mass spectrometer. In each case a distinctive reaction rate for the first H/D exchange was observed, and exchanges of up to three deuterium atoms occurred with characteristic ion abundances that could be used to differentiate the isomers. O(6)-Methyl guanine, for example, showed only one slow H/D exchange with D2O, whereas l-methyl guanine exchanged two hydrogen atoms at a significantly faster rate. On comparison of the possible resonance structures of each protonated isomer with the experimental information about the number and rate of H/D exchanges observed, a reaction mechanism involving a concerted proton abstraction-deuterium cation donation was proposed.

Synthesis of electrophore-labeled oligonucleotides and characterization by matrix-assisted laser desorption/ionization mass spectrometry.

Recent work to apply mass spectrometric methods to DNA analysis has led to the attachment of an electrophore to an oligonucleotide primer, with the purpose of investigating whether the advantages of electron capture ionization (increased ionization efficiency, reduced fragmentation) could be extended to larger molecules, such as Sanger sequence ladders. The stability of the electrophore-modified primers under conditions encountered during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was investigated. Four different electrophore labels were successfully attached to the 5' terminus of a 17-base, single-stranded oligodeoxyribonucleotide sequencing primer. The attached electrophore tags are robust under conditions used for sample preparation and MALDI-MS, and little or no fragmentation resulting from loss of the electrophore was observed. While no sensitivity enhancement was observed for the electrophore-labeled DNA, mass spectrometric conditions are discussed under which the electrophore labels could enhance the detection of DNA sequencing ladders.

Biotesting of wastewater: a comparative study using the Salmonella and CHO assay systems.

Means to assess the toxicity of wastewaters are essential to implementing the Federal Clean Water Act. Health risk assessment based on single chemicals is limited by the number of chemicals that can be identified and to those chemicals for which toxicity data are available. Long-term whole animal tests on large numbers of wastewater samples are not practical. In this study, two short-term tests, the Salmonella mutagenicity assay and the Chinese hamster ovary (CHO) cell assay for mutagenicity and cytotoxicity, were evaluated as potentially useful biomonitors of wastewaters. Standard assay protocols were modified to allow testing of up to 2.5 and 3.4 ml of unconcentrated water in the bacterial and mammalian cell tests, respectively. Cytotoxicity and mutagenicity were detected in some unconcentrated wastewater samples using these modifications. Data on eight wastewater samples, representing five different sites, indicated that the Salmonella test is the more sensitive indicator of mutagenic activity in those samples, whereas the CHO test is a sensitive indicator of the presence of cytotoxic components. Wastewater concentrates, prepared by adsorption onto XAD-2 and "blue cotton," were compared in the two bioassays. In a single concentrate, the two short-term tests detected distinctly different mutagens. Advantages of using the CHO-AS52 cell line instead of the CHO-K1BH4 line for detecting wastewater mutagens were indicated. This study illustrates the complementary use of multiple bioassays and concentration methods to detect and characterize toxic components in wastewater.

Toxic and teratogenic effects of chemical class fractions of a coal-gasification electrostatic precipitator tar.

Dimethyl sulfoxide slurries of a coal gasifier electrostatic precipitator tar and its chemical class fractions were assayed for their toxicity and teratogenicity using early embryos of the frog Xenopus laevis. Of the 5 tar fractions the ether-soluble base and polyaromatic were found to be the most teratogenic and the ether-soluble acid and ether-soluble base were the most toxic. The teratogenic effects of the raw tar suggest synergism. The toxic effects to newly metamorphosed froglets is 1-2 orders of magnitude less than those observed for embryos. Chemical analysis shows dihydroxybenzenes and organonitrogen compounds to be the major components of the acid and base fractions, respectively. The neutral fractions contain mainly alkyl-substituted two-ring hydrocarbons.

Rapid analysis of animal drug residues by microcolumn solid-phase extraction and thermal desorption-ion trap mass spectrometry.

A new approach was developed for the rapid and quantitative determination of an anthelmintic drug, phenothiazine, in milk. The technique involves a simple extraction procedure using a C18 microcolumn disc, followed by thermal desorption of the analyte from the disc directly into an ion trap mass spectrometer. The compounds are selectively ionized by isobutane chemical ionization and detected by tandem mass spectrometry. With this approach, 10 ppb detection limits were achieved with as little as 100 microL milk and only 10 min of analysis time. This approach was used to analyze samples of milk taken from a cow administered a one-time therapeutic dose of phenothiazine. The target compound could be detected at 56 h post-dosage, corresponding to a concentration of 30 ppb.

Rapid extraction and structural characterization of biomolecules in agarose gels by laser desorption Fourier transform mass spectrometry.

A method originally developed for the extraction of biomolecules from agarose gel slices has been utilized as a rapid means of isolating biological compounds from gels for subsequent structural characterization by matrix-assisted last desorption-ionization Fourier transform mass spectrometry (MALDI/FTMS). This "freeze-squeeze" extraction method involves pressure extrusion of fluid from frozen gel slices and provides near 50% recovery of analyte in less than 5 min. Experiments were directed at examining the recovery efficiency of the extraction method using 14C-labeled adenosine monophosphate and investigating the effect of high buffer concentrations on the laser desorption mass spectra. When coupled with this extraction technique, MALDI/FTMS can be used to detect and identify biomolecules at the low picomole level in agarose gel slices. The accurate mass measurements and MS/MS capabilities of the FTMS were exploited to provide detailed structural information at the isomeric level for oligonucleotides electrophoresed into agarose gels.

Characterization of photo-induced pyrimidine cyclobutane dimers by laser desorption Fourier transform mass spectrometry.

Laser desorption Fourier transform mass spectrometry was used to characterize the cis-syn cyclobutane photodimers of uracil-uracil, uracil-thymine and thymine-thymine. This soft ionization technique generated [M-H]- ions as well as some fragment ions. Investigation of the laser desorption process indicated that gas-phase dimerization reactions do not occur for pyrimidine monomers and dimers under these experimental conditions. Collisional dissociation of the [M-H]- ions provided structural information for the pyrimidine rings of the dimers. The fragment ions observed in the collisional dissociation spectra of these cyclobutane dimers suggested rearrangement of the [M-H]- parent ions to a macrocycle prior to dissociation.

Determination of cocaine and heroin vapor pressures using commercial and illicit samples.

The establishment of drug vapor pressure reference values provides valuable information in the development of vapor sensing devices for drug detection. The purpose of this work was to determine equilibrium headspace vapor pressures for commercial and illicit drug samples for use in such applications. Samples of cocaine, both free base and hydrochloride forms, and heroin hydrochloride were evaluated. The procedure used to measure the vapor pressures was a modification of a previously published method. Vapor pressure values at 20 degrees C previously reported for cocaine free base range from 0.37 x 10(-7) to 1.20 x 10(-7) Torr. The vapor pressure value determined in this study was 2.96 x 10(-7) Torr. It is likely that the discrepancies are due to differences in experimental conditions, varying sources of samples, and uncertainty in the methodologies. When the values were compared for commercial (99% purity) and illicit (unknown purity) sources of cocaine free base, there were no statistical differences in the projected room temperature (20 degrees C) vapor pressure values. However, the commercial and illicit samples of cocaine hydrochloride did show statistical differences. Although no comparison was made with the vapor pressure values for a pure, commercial sample, the vapor pressures of heroin hydrochloride (81% purity) at various temperatures were determined using the method developed for cocaine and are reported in this paper.

Matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry of oligodeoxyribonucleotides.

Conditions for the matrix-assisted laser desorption/ionization (MALDI) of oligodeoxyribonucleotides at 355 nm, developed using a 3-Tesla Fourier-transform ion cyclotron resonance mass spectrometer (FTMS), are reported. Efficient ion trapping and matrix selection are critical to the desorption and detection of oligonucleotides by FTMS. The achievable upper mass limit for the MALDI-FTMS of biomolecules on our 3-Tesla system has been extended from approximately 2 kDa to 6 kDa through the use of pulsed-trapping-plate ion deceleration techniques. By implementing the deceleration techniques, molecular ions for bovine insulin (MW = 5733.5), an oligodeoxythymidylic acid, pd[T]10 (MW = 3060.0), and a mixed-base 12-mer (MW = 3611.5) have been measured. For the analysis of oligonucleotides by FTMS, selection of an appropriate MALDI matrix is essential for the generation of [M-H]- ions. 3-Hydroxypicolinic acid provides a significant improvement over 2,5-dihydroxybenzoic acid for production of deprotonated molecules particularly for mixed-base oligomers. MALDI studies using FTMS have been duplicated using a newly constructed time-of-flight mass spectrometer (TOFMS) and oligonucleotide fragmentation on the TOFMS is reduced relative to that observed by FTMS. This may be a consequence of the longer times (milliseconds) required for FTMS detection.

Analysis of modified oligonucleotides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied to the structural characterization of modified oligodeoxyribonucleotide 4-, 6-, and 11-mers. Each oligonucleotide contained one modified base, either an O6-methyl-substituted guanine, an N6-(10R)-trans-opened benzo[a]pyrenediol epoxide adduct of adenine, or an N2-(R)-styrene oxide adduct of guanine. 3-Hydroxypicolinic acid was used as the MALDI matrix for molecular weight and purity determinations, while either 2,5-dihydroxybenzoic acid (DHBA) or an anthranilic/nicotinic acid (AA/NA) mixture was used to induce fragmentation for the production of structurally significant fragment ions. For the 4- and 6-mers, the oligonucleotide sequence could be obtained from the direct AA/NA or DHBA spectra. Sequence information was also obtained by inserting a time delay between the laser desorption event and ion detection to permit metastable decomposition. For the 11-mers, high-mass sequence ions were not detected. Although similar sequence ions were observed in both the positive and the negative ion mass spectra, more fragmentation was generally observed in the positive ion mode. In the positive ion mode, modified base fragment ions were observed when DHBA was used, and these fragments were examined using accurate mass measurements, collisionally induced dissociations, and ion-molecule reactions to characterize the modified base. MALDI-FTMS signals from one sample application can be used for the measurement of hundreds of spectra. The direct MALDI-FT mass spectra show matrix-dependent, structurally informative fragments, and CID experiments can be implemented using low-picomole sample quantities.

Detection of bacterial DNA polymerase chain reaction products by matrix-assisted laser desorption/ionization mass spectrometry.

Accurate monitoring and identification of Legionella species, the causative agents of Legionnaires' and other diseases, in environmental water sources is an important public health issue. Traditional culture methods often lack the sensitivity and specificity that can be attained using the polymerase chain reaction (PCR) to amplify targeted regions of the bacterial genome. Matrix-assisted laser desorption/ionization combined with time-of-flight (MALDI-TOF) mass spectrometry is shown to be useful for detection of 108- and 168-base PCR products specific to Legionella. A rapid purification aimed at removal of salts and unreacted primers is demonstrated. The addition of a synthetic DNA 20-mer to the MALDI sample facilitates aiming the laser at a favorable spot on the sample probe from which the PCR products can be detected.

Embryotoxic and teratogenic effects of aqueous extracts of tar from a coal gasification electrostatic precipitator.

Aqueous extracts of tar from a coal gasification electrostatic precipitator were tested for its toxic and teratogenic potential in vitro on embryos of the amphibian Xenopus laevis. The 96-h LC50 and EC50 were determined to be 0.83% and 0.48%, respectively. The developmental stage of normal-appearing exposed embryos is not affected by increasing concentrations of the extract. Embryo growth, however, is significantly reduced at concentrations as low as 0.25%. Motility and pigmentation were effectively reduced relative to controls by extract concentrations of 0.5% and greater. Exposed embryos are shorter and stockier than controls. Malformations of head, eyes, viscera, and spine are common, and cartilage formation is abnormal. The epidermis is often hyperplastic, and large blisters occur over the somatic surface. The severity of abnormal development is directly related to the concentration of the toxicant to which the embryos are exposed. Chemical analysis shows that the aqueous extracts contain phenols, furans, monoaromatic and diaromatic hydrocarbons, and mono- and diazaarenes and/or monoaromatic amines.

Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection.

Screening of mutant mice for subtle phenotypes requires sensitive, high-throughput analyses of sentinel proteins in functional pathways. The cytokine TNF-alpha is upregulated during inflammatory reactions associated with autoimmune diseases. We have developed a method to monitor the concentration of TNF-alpha under physiological conditions. TNF-alpha is captured, purified, and concentrated using monoclonal antibody-coated microbeads. The capture is efficient (> 80%) and can be used in the concentration range < 100 pg/mL to > 50 ng/mL, as determined by detection of 125I-labeled TNF-alpha. The bead capture of TNF-alpha can be combined with direct detection by MALDI-MS for sample concentrations of > 10 ng/mL. TNF-alpha can be captured and detected from diluted mouse serum, with minimal interferences observed in the MALDI spectrum. This method is adaptable to high-throughput sample handling with microfluidic devices and automated mass spectrometric analysis.

Structural characterization of polycyclic aromatic hydrocarbon dihydrodiol epoxide DNA adducts using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied for the structural characterization of four polycyclic aromatic hydrocarbon dihydrodiol epoxide (PAHDE) adducts, including the 5,6-dimethylchrysene DE adduct of 2'-deoxyadenosine, the 5-methyl- and 5,6-dimethylchrysene DE adducts of 2'-deoxyguanosine, and the benzo[a]pyrene-DE adduct of 2'-deoxyguanosyl 3'-phosphate. Measurement of positive and negative ion mass spectra, accurate mass determinations, and CID experiments were carried out using 10-40 ng (20-70 pmol) of sample. An evaluation of five MALDI matrices showed that matrix selection can be used to control the degree of analyte fragmentation. Three MALDI matrices commonly used for the analysis of proteins (sinapinic acid, ferulic acid, 2,5-dihydroxybenzoic acid) gave positive ion adduct mass spectra showing protonated or sodiated molecular ions accompanied by abundant, structurally informative fragment ions. Fragmentation was significantly reduced when working with two matrices used for oligonucleotide analysis (an anthranilic-nicotinic acid mixture and 3-hydroxypicolinic acid). Using the CID capabilities of FTMS, isolation and activation of the MALDI-produced ions was used to provide additional structural information. While characteristic negative ions were not detected for the adenosyladduct, the guanosyl and guanosyl 3'-phosphate adducts gave [M-H]- ions when the anthranilic-nicotinic acid matrix mixture was used. The guanosyl adducts also showed [M-H-2H2O]- fragments. Compared with FAB or FAB-MS/MS for the analysis of underivatized PAH-DE adducts, MALDI-FTMS signals are long-lived, the direct MALDI-FT mass spectra show more structurally informative fragments, and accurate mass and CID experiments require lower sample quantities.

Improving spot homogeneity by using polymer substrates in matrix-assisted laser desorption/ionization mass spectrometry of oligonucleotides.

We describe a method for improving the homogeneity of MALDI samples prepared for analysis of small, single-stranded oligonucleotides using the widely used DNA matrix system, 3-hydroxypicolinic acid/picolinic acid/ ammonium citrate. This matrix system typically produces large crystals around the rim of the dried sample and requires tedious searching of this rim with the laser. However, when a substrate is prepared using both Nafion and a hydrophilic, high-molecular-weight polymer, such as linear polyacrylamide, linear poly(ethylene oxide), or methyl cellulose, oligonucleotide-doped matrix crystals tend to be smaller and more uniformly distributed across the entire spot, thus decreasing the time that is required for locating a usable signal. In addition to MALDI characterization of the spatial distribution of "sweet spots," fluorescence microscopy allows for imaging dye-labeled DNA in dried MALDI spots. The mechanism of enhanced uniformity may involve increased viscosity in the MALDI sample droplet due to partial solubilization of the substrate by the MALDI sample solvent as well as partitioning of the matrix or DNA between the solvent and the undissolved portion of the polymer substrate.

MALDI-TOF analysis of polymerase chain reaction products from methanotrophic bacteria.

Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity. The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry. A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis. A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection. No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species. The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites.

The distribution of dermal tumorigens in coal liquids: relationship of tumorigenicity and microbial mutagenicity.

Polycyclic aromatic hydrocarbons and/or their pyrolle derivatives were found to be the primary contributors to the skin tumorigenicity of the neutral fractions of two coal oils. Mutagenicity of the neutral fraction in Salmonella test strains was found to be due primarily to polycyclic aromatics containing polar substituents. Thus, the chemical classes responsible for skin tumorigenicity differ from those responsible for mutagenicity.

A naturally occurring pyrimidodiazepine in Drosophila: chemical and spectral properties and relationship to drosopterin.

The structure of an intermediate, in drosopterin biosynthesis, as 6-acetylpyrimidodiazepine has been confirmed by high-resolution mass spectra, 13C NMR, chemical ionization mass spectra, and chemical properties. A trivial name of 6-acetylhomopterin is suggested and should replace the term "quench spot" used heretofore. The structure of drosopterin includes, in part, a pyrimidodiazepine, a compound that consists of a fused six- and seven-membered heterocyclic ring system. Earlier studies demonstrated that 6-acetylhomopterin strongly stimulated the enzymatic synthesis of drosopterin and related eye pigments by preparations from Drosophila. The occurrence in nature is quite limited for diazepines; drosopterin and homopterin are the first examples in eukaryotes.

Applications of mass spectrometry to DNA sequencing.

The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.