Direct mass spectrometric analysis of Bacillus spores

Spores from the Bacillus species, B. cereus, B. anthracis, B. thuringensis, B. lichenformis, B. globigi, and B. subtilis, were examined by direct probe mass spectrometry using electron ionization (EI) and positive and negative chemical ionization (CI). Molecular ions from free fatty acids and nucleic acids were observed in the 70eV spectra as were fragments from glycerides. Spectra obtained with isobutane positive chemical ionization (CI(+)) were dominated by ions associated with pyranose compounds such as N-acetylglucosamine (NAG). Unlike the positive ion spectra, the negative ion spectra of the spores were very simple and contained few peaks. The M(-.) ion from dipicolinic acid (DPA) was the base peak in the negative ion spectra of all spore species except those from B. lichenformis. The negative ion of DPA produced such a strong signal that 10(8) colony forming units (CFUs) of B. cereus spores could be detected directly in 0.5 g of ground rice. Principal component analysis (PCA) of the spectra revealed that only CI(+) spectra contained differences that could be used to identify the spectra by species. Differentiation of the CI(+) spectra by PCA was attributed to variances in the peaks associated with the bacterial polymer poly(3-hydroxybutyrate) (PHB) and NAG. Similar differences in PHB and NAG peaks were detected in the CI(+) spectra of a suite of vegetative Bacillus stains grown with various media.     

Raman spectra of dipicolinic acid in crystalline and liquid environments

Raman spectra of dipicolinic acid (DPA) are important for detection of bacterial spores, since DPA and its salts present one of their major components. The implementation of a deeply cooled CCD camera in combination with pulsed excitation at 532 nm allowed measuring well-resolved Raman spectra of the DPA in different forms. Powder preparations, crystals grown from saturated solutions and aqueous solutions of the DPA were studied. The spectral features in different environments and comparison with the spectra obtained by other methods are discussed.     

Pyrolysis mass spectrometry of terephthalate polyesters using negative ionization

The usual method of studying thermal degradation mechanisms of polymers in vacuo is to use electron ionization pyrolysis mass spectrometry. This can lead to mass spectral fragmentation from the 70 eV electrons used. Low energy electrons (10–15 eV) produce a low abundance of positive ions. However, if a molecule is prone to capture a thermal energy electron, then negative ions are produced in high abundance. This report describes the negative ion pyrolysis mass spectrometry of polyethylene terephthalate and polybutylene terephthalate.     

EFFECT OF DIPICOLINIC ACID ON THE ULTRAVIOLET RADIATION RESISTANCE OF BACILLUS CEREUS SPORES

Abstract— The ultraviolet radiation (UV) resistance of B. cereus spores was shown to depend on their content of dipicolinic acid (DPA). Wild-type spores with decreasing amounts of DPA exhibited increased UV resistance. Similarly, spores devoid of DPA (DPA-minus), produced by a mutant strain of B. cereus unable to synthesize DPA, were more resistant to UV than mutant spores (DPA-plus) produced in the presence of exogenously supplied DPA. Resistance of both the wild type and mutant strains to ionizing radiation, however, was unaffected by DPA content. Comparison of the resistance of DPA-minus and DPA-plus mutant spores to UV of various wavelengths showed that the greater sensitivity of the latter DPA-plus spores appeared at wavelengths corresponding to the region of the first molecular absorption band of the calcium chelate of DPA. In the wild type and mutant, thymine photoproducts were produced at a greater rate and to a greater extent in spores with high levels of DPA than in spores with low DPA.
The data indicate that DPA transfers energy to DN A in vivo , which leads to the conclusion that DPA occurs in the spore protoplast.     

In situ analysis of agrochemical residues on fruit using ambient ionization on a handheld mass spectrometer

We describe a rapid in situ method for detecting agrochemicals on the surface or in the tissue of fruit using a portable mass spectrometer equipped with an ambient ionization source. Two such ionization methods, low temperature plasma (LTP) and paper spray (PS), were employed in experiments performed at a local grocery store. LTP was used to detect diphenylamine (DPA) directly from the skin of apples in the store and those treated after harvest with DPA were recognized by MS and MS/MS. These data therefore allowed ready distinction between organic and non-organic apples. DPA was also found within the internal tissue of purchased apples and its distribution was mapped using LTP. Similarly, thiabendazole residues were detected on the skin of treated oranges in a grocery store experiment in which paper spray was performed by wiping the orange surface with a moist commercial lens wipe and then applying a high voltage to ionize the chemicals directly from the wipe. The handheld mass spectrometer used in these measurements is capable of performing several stages of tandem mass spectrometry (up to MS(5)); the compounds on the fruit were identified by their MS/MS fragmentation patterns. Protonated DPA (m/z 170) produced a characteristic MS(2) fragment ion at m/z 92, while thiabendazole was identified by MS(3) using precursor to fragment ion transitions m/z 202 →m/z 175 →m/z 131. These particular examples exemplify the power of in situ analysis of complex samples using ambient ionization and handheld mass spectrometers.     

紫外光度法间接测定脱脂乳中的芽孢数量

为了有效控制乳制品生产过程、保证质量,克服芽孢杆菌传统计数方法周期长的缺点,本实验通过分光光度法检测芽孢中特异性物质2,6-吡啶二羧酸(DPA)。根据DPA浓度与芽孢数量的关系,建立了快速检测脱脂乳中芽孢数量的方法。本研究对脱脂乳及其芽孢处理、DPA释放及采用紫外分光光度法检测做了研究。通过准确度和加标回收率检验,建立DPA含量与芽孢数的线性关系,回归方程为y=0.2084x-0.9363,r=0.6998,经验证可有效估测脱脂乳中芽孢数量(P<0.05)。     

Relevance of analytical pyrolysis studies to biomass conversion

The principles and methods of soft ionization mass spectrometry in combination with pyrolysis of macromolecules are outlined. Essential features of the newly developed techniques are the extension of the recorded mass range and the almost exclusive formation of molecular ions of the pyrolyzates. Using field ionization and field desorption mass spectrometry at low and high mass resolution, with electrical and photographic detection, pyrolysis products of biomass were analyzed for the first time. The results of flash pyrolysis by Curie-point heating and thermally programmed degradation of biopolymers are compared.The main topic is the evaluation of the methodology for time- and temperature-resolved pyrolysis. The thermograms and pyrolysis mass spectra obtained enable the study of pyrolysis reactions and the chemical fingerprinting of complex biological matter. The kinetics for the devolatilization of individual chemical species or classes of compounds can be monitored. Curie-point pyrolysis of biopolymers such as kappa-carrageenan and time-programmed degradation of cellulose and lignin are reported. Furthermore, preliminary investigations of pine wood and coal illustrate the potential of the introduced methods.     

Thermal degradation of spruce needles studied by time-resolved mass spectrometry and multivariate data analysis

In a study related to the impact of air pollution on forests, needles from a healthy and a severely damaged Norway spruce tree were analysed by temperature-programmed pyrolysis/field ionization (FI) mass spectrometry. Dried and pulverized spruce needles were heated at a rate of 0.6°C s^?1 to 450°C in the high vacuum of a FI ion source. Over 100 mass spectra were recorded electrically during each analysis. From each mass spectrum, average molecular weights of the pyrolysis products were calculated; their variation with pyrolysis temperature is discussed. The mass spectra in the range m/z 100–600 are used to calculate partial weight-loss curves. The FI mass spectra are evaluated by principal component analysis and factor rotation. The three-factor spectra based on loadings of the rotated principal components show typical FI signals which are produced during pyrolysis at low, medium and high temperatures. These signal patterns are interpreted as molecular ions of thermally stable, relatively volatile plant constituents and molecular ions of thermal degradation products derived from the thermolysis of carbohydrates, lignin and other biopolymers which occur in conifer needles. Medium- and high-temperature products of lignin can be distinguished. Principal component scores can be used to simulate the appearance of single FI signals, i.e., pyrolysis products. The evaluation of time-resolved pyrolysis and soft ionization mass spectrometric data from a single sample by principal component analysis and factor rotation appears to be suitable for characterization of the major chemical components and their thermal behaviour in complex biological samples.     

The three-dimensional structure of humic substances and soil organic matter studied by computational analytical chemistry

A novel three-dimensional structural concept for humic substances and soil organic matter (SOM) is proposed which is based on previously published, comprehensive investigations combining geochemical, wetchemical, biochemical, spectroscopic, agricultural and ecological data with analytical pyrolysis. Direct, temperature-programmed pyrolysis in the ion-source of the mass spectrometer and soft ionization in very high electric fields (Py-FIMS) and Curie-point pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) were the main applied thermal methods. Emphasis is laid on molecular modelling and geometry optimization of complex, polydisperse structures of biomacromolecules using modern PC software (HyperChem^®). Trapping and binding of atrazine in an organo-mineral complex is introduced as a first example of simulation experiments for soil processes at atomic level (nanochemistry). Future applications of semi-empirical calculations and molecular dynamics in pyrolysis studies are outlined.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

Comparison of EI and metastable atom bombardment ionization for the identification of polyurethane thermal degradation products

Polyurethanes are widely used in the manufacture of commercial products such as foams and paints. During combustion, these polymers can generate isocyanates, which induce adverse health effects. Polymer pyrolysis (Py) hyphenated with mass spectrometry (MS) allows the investigation of polymer thermal degradation over time/temperature. A diphenylmethanediisocyanate (MDI) polyurethane foam was analyzed with electron ionization (EI) and metastable atom bombardment (MAB) ionization at a pyrolysis temperature of 400 °C. The recently introduced MAB ionization source uses discrete energy stored in metastable atoms of gases to ionize the analytes. This characteristic allows modulation of the ionization energy by simply changing the ionization gas. The extensive fragmentation of molecular ions observed using EI 70 eV is not totally eliminated with EI 10 eV. However, only molecular ions are observed with MAB using N₂ as the ionization gas. Temperature gradients were used to separate the products generated during the thermal degradation of a 1,6-hexamethylenediisocyanate (HDI) polyurethane paint. The analysis of mass spectra was facilitated owing to a selective desorption of pyrolysis products. Furthermore, changing the MAB ionization gas allows elucidation of the structure of the pyrolysis products by controlling the extent of their fragmentation. During these experiments, isocyanic acid, methyleneisocyanate, ethyleneisocyanate, propylisocyanate and butylisocyanate were detected.     

Characterization of Aspergillus spores by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The intact fungal spores of several strains of four Aspergillus species, Aspergillus flavus, A. oryzae, A. parasiticus, and A. sojae, were directly analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Very simple MALDI mass spectra are obtained by directly mixing spores with a matrix such as alpha-cyano-4-hydroxycinnamic acid or sinapinic acid. The mass spectra are obtained from the ablation of cell walls of spores owing to the acidity of the matrix solution. The MALDI results show that aflatoxigenic strains and non-aflatoxigenic strains have different mass peak profiles. Furthermore, the MALDI results of non-aflatoxigenic A. flavus and A. parasiticus spores resemble those of the closely related A. oryzae and A. sojae spores, respectively.     

Rapid characterization of biomaterials by field ionization

The direct application of field ionization to complex biomaterials is described. Volatiles are characterized by gas chromatography mass spectrometry. Complex involatile materials are investigated by thermal degradation in high vacuum. The methodology and typical analytical features of pyrolysis of biopolymers and soft ionization of their pyrolysates in the high electric field are described. The combination of direct, in-source pyrolysis mass spectrometry and pyrolysis gas chromatography mass spectrometry is used in two steps. Firstly, for fast profiling of the complex materials and, secondly, for identification of significant products of the controlled thermal degradation process. In particular, temperature programmed/time-resolved pyrolysis field ionization mass spectrometry has been shown to be an efficient analytical tool as demonstrated for typical examples such as chitin, cellulose, hemicellulose, lignin, wood, peat and coal. Recent results on foodstuffs such as coffee, chocolate, tea and biscuits illustrate the potential of the combined methods for routine work.     

Dipicolinic acid (DPA) assay revisited and appraised for spore detection

Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests.     

Characterization of intact Penicillium spores by matrix-assisted laser desorption/ionization mass spectrometry

The fungal spores of Penicillium expansum, P. chrysogenum, P. citrinum, P. digitatum, P. italicum, and P. pinophilum were characterized by using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOFMS). These fungal spores are frequently found in grain and fruit. The mass spectra of these six species were directly obtained from the intact spores without any pretreatment. The results obtained indicate that 2,5-dihydroxybenzoic acid and sinapinic acid are suitable matrices for the analysis of Penicillium spores. Characteristic ions representing the different species were obtained with sufficiently high reproducibility that these ions can be employed to identify the different fungal species. On the basis of these characteristic ions obtained from these authentic Penicillium spores, the approach was applied to characterize the fungal species contaminating the surfaces of fruit. It was demonstrated that the fungal spores directly scratched from the surfaces of fruit contaminated by unknown fungi can be rapidly identified using MALDI-TOFMS analysis without any tedious pretreatment.     

Pyrolysis–gas chromatography mass spectrometry and field ionization mass spectrometry of polyquinones

Low- and high-molecular mass thermal decomposition products of five polyquinones with different linking aromatic structures have been analyzed by pyrolysis–gas chromatography and by direct (in-source) pyrolysis–field ionization mass spectrometry. The quantity of carboxyl groups present in the polymer is obtained by the amounts of carbon dioxide found by pyrolysis–gas chromatography. Assuming a radical thermal decomposition mechanism the distribution of ketoacidic and quinonoid segments along the macromolecular ladder could be estimated from the high-molecular mass products measured by pyrolysis–field ionization mass spectrometry. A random distribution of the two different segments was found for polyquinones with biphenylene and dibenzofuran subunits, while a structure built up of blocks of two or more identical segments was obtained for polyquinones with dibenzothiophene and diphenylmethane subunits. At the same time the anomalous structural moieties in the polyquinone ladders are also clarified with the help of the identification of the unexpected pyroysis products. Oxidated and bis-dibenzothiophene and bis-diphenylmethane subunits were found. The observed temperature dependence for the appearances of the thermal degradation products indicates that condensation and elimination reactions are taking place under the described pyrolysis conditions. Condensation in the ketoacidic segments forming new quinonoid segments proved to be important in the polymer which was a 100% poly(ketoacid), but negligible in the polyquinones containing ketoacidic segments up to 60%.     

Structural characterization and sequence distributions of polysiloxanes using pyrolysis MS/MS

Novel polysiloxanes, with 4-(dialkylamino)pyridine substituents, are characterized by pyrolysis tandem mass spectrometry. These polymers form abundant cyclic oligomeric ions under both desorption electron ionization (DEI) and desorption chemical ionization (DCI) conditions. Product MS/MS spectra of the cyclic ions reveal characteristic fragmentations under low energy collision activated dissociation. Protonated cyclic oligomers higher than the pentamer are mainly due to the proton bound dimers of lower oligomeric units. The cyclic oligomers are shown to have proton affinities greater than 1000 kJ/mole. It is proposed that thermal depolymerization occurs through an intramolecular siloxane bond rearrangement, which is in agreement with a previously proposed "loop mechanism". Markovian statistical calculations are applied to the DCI mass spectral data in order to determine the sequence distribution of siloxane copolymers. Application of this method show that the monomers in the copolymers examined are non-randomly distributed.     

Complete sequences of small acid-soluble proteins from Bacillus globigii

Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry. The proteins were extracted from spores with 1 M HCl. Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment. Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (gamma-SASP). De novo peptide sequencing was used to determine the protein sequences. The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus. B. globigii is used in many field tests as a surrogate for B. anthracis. Thus complete SASP sequences from B. globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species.     

Pyrolysis field ionization mass spectrometry of carbohydrates: Part b: Polysaccharides

The application of pyrolysis in combination with field ionization (FI) mass spectrometry for the characterization and identification of polysaccharides is reported. Polysaccharides such as xylan, agarose and alginic acid, which contain monomer subunits of different elemental composition, can be differentiated in a straightforward manner by the field ionization spectra of their Curie-point pyrolysates. Polysaccharides with hexosyl subunits, such as cellulose, galactan, laminaran and mannan, were pyrolysed by Curie-point pyrolysis and show photographically recorded FI spectra which differ in the relative heights of their pyrolysis peaks. Characteristic pyrolysis products are formed, which can be identified or assigned structures on the basis of accurate mass measurements, direct isotopic determination and by analogy with established chemical procedures and mechanisms.Oven pyrolysis of polysaccharides combined with electrical detection of the FI spectra at low mass resolution gives a higher sensitivity and better reproducibility for all peaks over the whole mass range. From sample amounts of about 40 μg, spectra are obtained by raising the oven temperature by 0.4°C/s. Utilizing repetitive magnetic scanning, registration and signal processing by the data system, the standard deviation of the peak heights for five repeated measurements is about 10%. Accumulation of about 30 spectra in a limited mass range on a multi-channel analyser gives results which vary by about 2–3% on average, despite a lower sample consumption (20–30 μg). Oven pyrolysis between 250 and 400°C yields significant differences in the spectra of differently linked mannans and allows an unequivocal differentiation of these isomers. Following FI, field desorption (FD) spectra were obtained from pyrolysis products condensed on the emitter surface by heating of the emitter wire between 10 and 30 mA. The cations of alkali metals, such as Na*, K* and Cs*, can be registered in this way. Most interesting is the detection of the molecular ions of monomer and oligomer subunits of the polysaccharides as complementary analytical information in the FD mode. Obviously, condensation of these neutral, thermal products on the emitter surface occurs without field ionization and desorption is initiated by supply of thermal energy to the adsorbed sample layer.     

Mass spectrometric behavior of levoglucosan under different ionization conditions and implications for studies of cellulose pyrolysis

Pyrolysis-mass spectrometric studies of cellulose indicate low abundances of levoglucosan in the product spectrum compared to the yield values determined in more conventional types of pyrolysis studies. To examine the reason for these conflicting observation, levoglucosan was examined under different ion source conditions and ionization modes to ascertain the relative contributions of thermal degradation and ionization fragmention to the low abundances of the levoglucosan molecular ion. Low-energy electron ionization using conventional sample volatilization and molecular-beam sampling is compared to chemical ionization using methane, isobutane, and ammonia as reagent gases, and to field ionization and desorption. The mass spectrometric fragmentation patterns under the various systems indicate that studies of cellulose pyrolysis underestimate the amount of levoglucosan formed due to ionization fragmentation and thermal rearrangement reactions in the ion source. Several peaks, including m/z 126 and 144, are dominated by the contribution from the fragmentation of levoglucosan.     

Using electrospray-assisted pyrolysis ionization/mass spectrometry for the rapid characterization of trace polar components in crude oil, amber, humic substances, and rubber samples

We describe the use of electrospray-assisted pyrolysis ionization/mass spectrometry (ESA-Py/MS) to selectively ionize trace polar compounds that coexist with large amounts of nonpolar hydrocarbons in crude oil, amber, humic substances, and rubber samples. Samples of different origins are distinguished rapidly by their positive ion ESA-Py mass spectra without prior separation or chemical pretreatment. During ESA-Py analysis, the samples in their solid or liquid states were pyrolyzed at 590, 630 or 940 degrees C using a commercial Curie-point pyrolysis probe. The gaseous pyrolysates were transferred into a glass reaction cell. The polar compounds (M) in the pyrolysates were then ionized by electrospray ionization (ESI), yielding protonated molecules (MH+). Although the major components of the pyrolysates are nonpolar hydrocarbons, their lack of functional groups that can receive a proton in the ESA-Py source results in no hydrocarbon ion signals being produced; thus, the positive ions detected in ESA-Py mass spectra all result from trace polar components in the pyrolysates.