Desorption/ionization fluence thresholds and improved mass spectral consistency measured using a flattop laser profile in the bioaerosol mass spectrometry of single Bacillus endospores

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Mass spectra of individual Bacillus endospores were measured with a bipolar aerosol time-of-flight mass spectrometer in which molecular desorption and ionization were produced using a single laser pulse from a Q-switched, frequency-quadrupled Nd:YAG laser that was modified to have an approximately flattop profile. The flattened laser profile allowed the minimum fluence required to desorb and ionize significant numbers of ions from single aerosol particles to be determined. For Bacillus spores, this threshold had a mean value of approximately 1 nJ/microm(2) (0.1 J/cm(2)). Thresholds for individual spores, however, could apparently deviate by 20% or more from the mean. Threshold distributions for clumps of MS2 bacteriophage and bovine serum albumin were subsequently determined. Finally, the flattened profile was observed to increase the reproducibility of single-spore mass spectra. This is consistent with the general conclusions of our earlier paper on the fluence dependence of single-spore mass spectra and is particularly significant because it is expected to enable more robust differentiation and identification of single bioaerosol particles.     

Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var. niger were obtained in a bipolar aerosol time-of-flight mass spectrometer using a pulsed 266-nm laser for molecular desorption and ionization. Spectra from single spores collected at an average fluence of approximately 0.1 J/cm2 frequently contain prominent peaks attributed to arginine, dipicolinic acid, and glutamic acid, but the shot-to-shot (spore-to-spore) variability in the data may make it difficult to consistently distinguish closely related Bacillus species with an automated routine. Fortunately, a study of the laser power dependence of the mass spectra reveals clear trends and a finite number of "spectral types" that span most of the variability. This, we will show, indicates that a significant fraction of the variability must be attributed to fluence variations in the profile of the laser beam.     

Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry

We have fully characterized the mass spectral signatures of individual Bacillus atrophaeus spores obtained using matrix-free laser desorption/ionization bioaerosol mass spectrometry (BAMS). Mass spectra of spores grown in unlabeled, 13C-labeled, and 15N-labeled growth media were used to determine the number of carbon and nitrogen atoms associated with each mass peak observed in mass spectra from positive and negative ions. To determine the parent ion structure associated with fragment ion peaks, the fragmentation patterns of several chemical standards were independently determined. Our results confirm prior assignments of dipicolinic acid, amino acids, and calcium complex ions made in the spore mass spectra. The identities of several previously unidentified mass peaks, key to the recognition of Bacillus spores by BAMS, have also been revealed. Specifically, a set of fragment peaks in the negative polarity is shown to be consistent with the fragmentation pattern of purine nucleobase-containing compounds. The identity of m/z = +74, a marker peak that helps discriminate B. atrophaeus from Bacillus thuringiensis spores grown in rich media is [N1C4H12]+. A probable precursor molecule for the [N1C4H12]+ ion observed in spore spectra is trimethylglycine (+N(CH3)3CH2COOH), which produces a m/z = +74 peak when ionized in the presence of dipicolinic acid. A clear assignment of all the mass peaks in the spectra from bacterial spores, as presented in this work, establishes their relationship to the spore chemical composition and facilitates the evaluation of the robustness of "marker" peaks. This is especially relevant for peaks that have been used to discriminate Bacillus spore species, B. thuringiensis and B. atrophaeus, in our previous studies.     

Detection of anthrax simulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores

Recent advances in the development of ultrasensitive micromechnical thermal detectors have led to the advent of novel subfemtojoule microcalorimetric spectoscopy (CalSpec). On the basis of principles of photothermal IR spectroscopy combined with efficient thermomechanical transduction, CalSpec provides acquisition of vibrational spectra of microscopic samples and absorbates. We use CalSpec as a method of identifying nanogram quantities of biological micro-organisms. Our studies focus on Bacillus subtilis and Bacillus cereus spores as simulants for Bacillus anthracis spores. Using CalSpec, we measured IR spectra of B. subtilis and B. cereus spores present on surfaces in nanogram quantities (approximately 100-1,000 spores). The spectra acquired in the wavelength range of 690-4000 cm(-1) (2.5-14.5 microm) contain information-rich vibrational signatures that reflect the different ratios of biochemical makeup of the micro-organisms. The distinctive features in the spectra obtained for the two types of microorganism can be used to distinguish between the spores of the Bacillus family. As compared with conventional IR and Fourier-transform IR microscopic spectroscopy techniques, the advantages of the present technique include significantly improved sensitivity (at least a full order of magnitude), absence of expensive IR detectors, and excellent potential for miniaturization.     

Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A novel approach to microbial detection using atmospheric pressure matrix-assisted laser desorption/ionization with an ion trap mass spectrometer to analyze whole cell bacteria is introduced. This new approach was tested with lyophilized spores and cultures of Bacillus globigii (BG) grown on agar media for 4 days or longer. At each stage of growth, it was found that biomarkers, identified as cyclic lipopeptides known as fengycin and surfactin, could be detected by pulsed ultraviolet laser irradiation of intact BG cells (approximately 5 mg) cocrystallized with alpha-cyano-4-hydroxycinnamic acid. Furthermore, definitive amino acid sequence information was obtained by performing tandem mass spectrometry on the precursor ions of the cyclic lipopeptides. The investigation was broadened to include the examination of aerosolized BG spores collected from the atmosphere and directly deposited onto double-sided tape. Subsequent analysis of the recovered spores resulted in the production of mass peaks consistent with fengycin. Other Bacillus species were analyzed for comparison and showed mass spectral peaks also identified as originating from various cyclic lipopeptides. Further studies were conducted using a pulsed infrared laser as the excitation source to analyze BG cells (approximately 5 mg) suspended in a matrix of 0.03 M ammonium citrate and glycerol resulting in the production of ions characteristic of fengycin and surfactin.     

Identification of bacterial spores using statistical analysis of Fourier transform infrared photoacoustic spectroscopy data

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) has been applied for the first time to the identification and speciation of bacterial spores. A total of forty specimens representing five strains of Bacillus spores (Bacillus subtilis ATCC 49760, Bacillus atrophaeus ATCC 49337, Bacillus subtilis 6051, Bacillus thuringiensis subsp. kurstaki, and Bacillus globigii Dugway) were analyzed. Spores were deposited, with minimal preparation, into the photoacoustic sample cup and their spectra recorded. Principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations were used on this spectral library to develop algorithms for step-wise classification at three levels: (1) bacterial/nonbacterial, (2) membership within the spore library, and (3) bacterial strain. Internal cross-validation studies on library spectra yielded classification success rates of 87% or better at each of these three levels. Analysis of fifteen blind samples, which included five samples of spores already in the spectral library, two samples of closely related Bacillus globigii 01 spores not in the library, and eight samples of nonbacterial materials, yielded 100% accuracy in distinguishing among bacterial/nonbacterial samples, membership in the library, and bacterial strains within the library.     

Direct from polyacrylamide gel infrared laser desorption/ionization

The direct combination of gel electrophoresis and infrared laser desorption/ionization time-of-flight mass spectrometry has been demonstrated. We present results for infrared laser desorption and ionization mass spectrometry of peptides and proteins directly from a polyacrylamide gel without the addition of a matrix. Analyte molecules up to 6 kDa were ionized directly from a vacuum-dried sodium dodecyl sulfate-polyacrylamide gel after electrophoretic separation. Mass spectra were obtained at the wavelength of 2.94 microm, which is consistent with IR absorption by N-H and O-H stretch vibrations of water and other constituents of the gel. A 5-nmol quantity of peptide or protein was loaded per gel slot, although it was possible to obtain mass spectra from a small fraction of the gel spot. This technique shows promise for the direct identification of both parent and fragment masses of proteins contained in polyacrylamide gels.     

Intensities of calcium dipicolinate and Bacillus subtilis spore Raman spectra excited with 244 nm light

Ultraviolet (UV) resonance Raman spectra of Bacillus subtilis endospores have been excited at 244 nm. Spectra can be interpreted in terms of contributions from calcium dipicolinate and nucleic acid components. Differences between spectra of spores and vegetative cells are very large and are due to the dominance of the dipicolinate features in the spore spectra. Because the DNA and RNA composition of B. subtilis spores is known and because the cross-sections of Raman bands belonging to DNA and RNA bases are known, it is possible to calculate resonance Raman spectral cross-sections for the spore Raman peaks associated with the nucleic acids. The cross-sections of peaks associated with calcium dipicolinate have been measured from aqueous solutions. Cross-section values of the dominant 1017 cm(-1) calcium dipicolinate peak measured from the Bacillus spores have been shown to be consistent with a calcium dipicolinate composition of ten percent or less by weight in the spores. It is suggested that spectral cross-sections of endospores excited at 244 nm can be estimated to be the sum of the cross-sections of the calcium dipicolinate, DNA, and RNA components of the spore. It appears that the peaks due to DNA and RNA can be used as an internal standard in the calculation of spore Raman peak cross-sections, and potentially the amount of calcium dipicolinate in spores. It is estimated on the basis of known nucleic acid base cross-sections that the most intense Raman band of the Bacillus subtilis spore spectra has a cross-section of no more than 4 x 10(-18) cm(2)/mol-sr.     

Cation selectivity of natural and synthetic ionophores probed with laser-induced liquid beam mass spectrometry

The novel laser desorption method laser-induced liquid beam ionization/desorption (LILBID) is applied to the mass spectrometric examination of selective ion binding by natural and synthetic ionophores in methanol solutions. The ions are desorbed from a liquid jet with an IR laser pulse and then extracted perpendicularly into a reflectron time-of-flight (RE-TOF) analyzer. LILBID studies on the natural ion carriers valinomycin and monensin A are presented, as well as those on the synthetic crown ethers 18-crown-6, diaza-18-crown-6, and benzo-15-crown-5. No fragment ions are detected, and the measured ion selectivity is in good qualitative agreement with published stability constants of the complexes. The observed specific recognition of silver ions by diaza-18-crown-6 can be rationalized by the principle of hard and soft acids and bases, which predicts stable complexes when the polarizabilities of Lewis acid and base are similar. Weak, noncovalent interactions like those in the sandwich complex between two benzo-15-crown-5 molecules with one potassium ion are detected with LILBID. Their preservation during the process of ion desorption depends on the laser intensity. A comparison with spectra obtained by using electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) shows that LILBID can potentially become a sensitive tool for the screening of weak but specific molecular interactions.     

Comparing Vacuum and Extreme Ultraviolet Radiation for Postionization of Laser Desorbed Neutrals from Bacterial Biofilms and Organic Fullerenes

Vacuum and extreme ultraviolet radiation from 8 - 24 eV generated at a synchrotron was used to postionize laser desorbed neutrals of antibiotic-treated biofilms and a modified fullerene using laser desorption postionization mass spectrometry (LDPI-MS). Results show detection of the parent ion, various fragments, and extracellular material from biofilms using LDPI-MS with both vacuum and extreme ultraviolet photons. Parent ions were observed for both cases, but extreme ultraviolet photons (16 - 24 eV) induced more fragmentation than vacuum ultraviolet (8 - 14 eV) photons.     

Stable isotope labeling of entire Bacillus atrophaeus spores and vegetative cells using bioaerosol mass spectrometry

Single vegetative cells and spores of Bacillus atrophaeus, formerly Bacillus subtilis var. niger, were analyzed using bioaerosol mass spectrometry. Key biomarkers were identified from organisms grown in 13C and 15N isotopically enriched media. Spore spectra contain peaks from dicipolinate and amino acids. The results indicate that compounds observed in the spectra correspond to material from the spore's core and not the exosporium. Standard compounds and mixtures were analyzed for comparison. The biomarkers for vegetative cells were clearly different from those of the spores, consisting mainly of phosphate clusters and amino acid fragments.     

Detection of native protein ions in aqueous solution under ambient conditions by electrospray laser desorption/ionization mass spectrometry

Liquid electrospray laser desorption/ionization (ELDI) mass spectrometry allows desorption and ionization of proteins directly from aqueous solutions and biological fluids under ambient conditions. Native protein ions such as those of myoglobin, cytochrome c, and hemoglobin were obtained. A droplet (ca. 5 microL) containing the protein molecules and micrometer-sized particles (e.g., carbon graphite powder) is irradiated with a pulsed UV laser. The laser energy adsorbed by the inert particles is transferred to the surrounding solvent and protein molecules, leading to their desorption; the desorbed gaseous molecules are then postionized within an electrospray (ESI) plume to generate the ESI-like protein ions. With the use of this technique, we detected only the protonated protein ions in various biological fluids (including human tears, cow milk, serum, and bacterial extracts) without interference from their corresponding sodiated or potassiated adduct ions. In addition, we rapidly quantified the levels of glycosylated hemoglobin present in drops of whole blood obtained from diabetic patients without the need of sample pretreatment.     

Aerosol-fluorescence spectrum analyzer: real-time measurement of emission spectra of airborne biological particles

We have assembled an aerosol-fluorescence spectrum analyzer (AFS), which can measure the fluorescence spectra and elastic scattering of airborne particles as they flow through a laser beam. The aerosols traverse a scattering cell where they are illuminated with intense (50 kW/cm(2)) light inside the cavity of an argon-ion laser operating at 488 nm. This AFS can obtain fluorescence spectra of individual dye-doped polystyrene microspheres as small as 0.5 μm in diameter. The spectra obtained from microspheres doped with pink and green-yellow dyes are clearly different. We have also detected the fluorescence spectra of airborne particles (although not single particles) made from various biological materials, e.g., Bacillus subtilis spores, B. anthrasis spores, riboflavin, and tree leaves. The AFS may be useful in detecting and characterizing airborne bacteria and other airborne particles of biological origin.     

Laser desorption from the surface of copper phthalocyanine films on silicon and cadmium sulfide

We have studied laser desorption mass spectra of copper phthalocyanine (CuPc) films deposited in vacuum onto single crystal silicon and cadmium sulfide substrates. The desorption was induced by 10-ns pulses of neodymium laser radiation (quantum energy, 2.34 eV) with an energy density E varied from 0.1 to 40 mJ/cm². It is established that laser radiation produces fragmentation of CuPc molecules and desorption of the fragments. The main fragments observed in the mass spectra are identified. The intensity of the main desorbed species has been studied as a function of the laser pulse energy density. The components of CdS substrates penetrate into the volume of deposited CuPc films in the form of Cd atoms and S₂ molecules.     

Laser desorption/ionization time-of-flight mass spectrometry on sol-gel-derived 2,5-dihydroxybenzoic acid film

This work presents a novel method for direct desorption/ ionization of analytes from sol-gel-derived film. 2,5-Dihydroxy benzoic acid (DHB), a common MALDI matrix, was incorporated into a sol-gel polymeric structure. The sol-gel-derived DHB thin film can assist the mass analysis of analytes by laser desorption/ionization, with a matrix interference-free background in the mass spectra. The sol-gel-derived film can function as an energy absorber during laser irradiation because it contains DHB molecules. Furthermore, laser irradiation with normal laser power (70-110 microJ) is not likely to generate any background ions from this sol-gel-DHB derived film. The samples were prepared straightforwardly. After a thin film was formed on a Parafilm membrane from the sol-gel-derived DHB solution coating, the sample solution was directly added to the top of the film, for laser desorption/ ionization mass analysis. The analyte signals were homogeneously obtained on the sol-gel-derived DHB film. Experimental results show that the optimum concentrations of DHB incorporated in the sol-gel solution were between 7,500 ppm and 10,000 ppm, providing a matrix interference-free background. Analytes, including small proteins, peptides, amino acids, and small organics, were used to demonstrate the effectiveness of the proposed method. However, a higher laser power (> 110 microJ) than normal was required to desorb small proteins from the sol-gel-derived DHB film. Therefore, a few matrix ions desorbed from the thin film were generated during laser irradiation. The detection limit for both small molecules and proteins, using this sol-gel-assisted laser desorption/ ionization (SGALDI) mass spectrometry (MS), was as low as 81 fmol. However, a mass spectrometer with cutoff-mass selection could detect 8.1 fmol of cytochrome c. The largest analyte observed by the SGALDI-MS in this study was myoglobin.     

Nonresonant MALDI of oligonucleotides: mechanism of ion desorption

Oligonucleotide ions have been detected using matrix-assisted laser desorption/ionization (MALDI) under nonresonant laser irradiation of the sample. When mass resolution was not limited by adduct attachment to the analyte ions, the nonresonant MALDI spectra demonstrated better resolution than the spectra acquired with resonant ultraviolet irradiation. We found that preparation of thin-film samples on absorbing substrate surfaces was critical for the success of NR-MALDI. The possible acoustic mechanisms of ion formation and desorption are discussed.     

Near-infrared surface-enhanced-Raman-scattering-mediated detection of single optically trapped bacterial spores

A novel methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants. This technique may be useful in many applications; most notably, development of novel detection schemes toward potentially harmful bacteria. This method would also be useful as an ancillary environmental monitoring system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally invasive medical applications). This unique detection scheme is based on the near-infrared (NIR) surface-enhanced Raman scattering (SERS) from single, optically trapped, bacterial spores. The SERS spectra of bacterial spores in aqueous media have been measured using SERS substrates based on approximately 60-nm-diameter gold colloids bound to 3-aminopropyltriethoxysilane derivatized glass. The light from a 787-nm laser diode was used to trap and manipulate as well as simultaneously excite the SERS of an individual bacterial spore. The collected SERS spectra were examined for uniqueness and the applicability of this technique for the strain discrimination of Bacillus stearothermophilus spores. Comparison of normal Raman and SERS spectra reveals not only an enhancement of the normal Raman spectral features but also the appearance of spectral features absent in the normal Raman spectrum.     

Fourier transform infrared reflectance microspectroscopy study of Bacillus subtilis engineered without dipicolinic acid: the contribution of calcium dipicolinate to the mid-infrared absorbance of Bacillus subtilis endospores

Mid-infrared spectra of spores of two strains of Bacillus subtilis, PS832 (wild-type) and FB122 (sleB spoVF), that are isogenic except for the two mutations in FB122 were obtained by Fourier transform infrared (FT-IR) reflectance microspectroscopy. The mutations in FB122 cause the spores of this strain to be devoid of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a biomarker characteristic of bacterial spores. Analysis of these two strains by difference spectroscopy revealed a spectrum similar to that of calcium dipicolinate (CaDPA), a chelate salt of DPA. This difference spectrum was compared to mid-infrared spectra of both DPA and CaDPA, and was attributed to CaDPA only. This is the first report known to the authors of a genetically engineered organism being used to identify the spectral contribution of a particular cellular component.     

Generation of highly charged peptide and protein ions by atmospheric pressure matrix-assisted infrared laser desorption/ionization ion trap mass spectrometry

We show that highly charged ions can be generated if a pulsed infrared laser and a glycerol matrix are employed for atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry with a quadrupole ion trap. Already for small peptides like bradykinin, doubly protonated ions form the most abundant analyte signal in the mass spectra. The center of the charge-state distribution increases with the size of the analyte. For example, insulin is detected with a most abundant ion signal corresponding to a charge state of four, whereas for cytochrome c, the 10 times protonated ion species produces the most intense signal. Myoglobin is observed with up to 13 charges. The high m/z ratios allow us to use the Paul trap for the detection of MALDI-generated protein ions that are, owing to their high molecular weight, not amenable in their singly protonated charge state. Formation of multiple charges critically depends on the addition of diluted acid to the analyte-matrix solution. Tandem mass spectra generated by collision-induced dissociation of doubly charged peptides are also presented. The findings allow speculations about the involvement of electrospray ionization processes in these MALDI experiments.     

Measured infrared optical cross sections for a variety of chemical and biological aerosol simulants

We conducted a series of spectral extinction measurements on a variety of aerosolized chemical and biological simulants over the spectral range 3-13 microm using conventional Fourier-transform IR (FTIR) aerosol spectroscopy. Samples consist of both aerosolized particulates and atomized liquids. Materials considered include Bacillus subtilis endospores, lyophilized ovalbumin, polyethylene glycol, dimethicone (SF-96), and three common background materials: kaolin clay (hydrated aluminum silicate), Arizona road dust (primarily SiO2), and diesel soot. Aerosol size distributions and mass density were measured simultaneously with the FTIR spectra. As a result, all optical parameters presented here are mass normalized, i.e., in square meters per gram. In an effort to establish the utility of using Mie theory to predict such parameters, we conducted a series of calculations. For materials in which the complex indices of refraction are known, e.g., silicone oil (SF-96) and kaolin, measured size distributions were convolved with Mie theory and the resultant spectral extinction calculated. Where there was good agreement between measured and calculated extinction spectra, absorption, total scattering, and backscatter were also calculated.