Title:
Strain-Level Bacterial Identification by CeO2-Catalyzed MALDI-TOF MS Fatty Acid Analysis and Comparison to Commercial Protein-Based Methods
Authors:
Cox CR, Jensen KR, Saichek NR, Voorhees KJ.
Goal:
We aimed to see if we could analyze bacteria colonies using CeO2 as a matrix in SALDI-MS and then use multivariate statistics to see if we could classify and identify the bacteria. We compared our method to a commercial method.
Link to Full Text:
https://doi.org/10.1038/srep10470
Abstract:
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid approach for clinical bacterial identification. However, current protein-based commercial bacterial ID methods fall short when differentiating closely related species/strains. To address this shortcoming, we employed CeO2-catalyzed fragmentation of lipids to produce fatty acids using the energy inherent to the MALDI laser as a novel alternative to protein profiling. Fatty acid profiles collected from Enterobacteriaceae, Acinetobacter, and Listeria using CeO2-catalyzed metal oxide laser ionization (MOLI MS), processed by principal component analysis, and validated by leave–one-out cross-validation (CV), showed 100% correct classification at the species level and 98% at the strain level. In comparison, protein profile data from the same bacteria yielded 32%, 54% and 67% mean species-level accuracy using two MALDI-TOF MS platforms, respectively. In addition, several pathogens were misidentified by protein profiling as non-pathogens and vice versa. These results suggest novel CeO2-catalyzed lipid fragmentation readily produced (i) taxonomically tractable fatty acid profiles by MOLI MS, (ii) highly accurate bacterial classification and (iii) consistent strain-level ID for bacteria that were routinely misidentified by protein-based methods.
Title:
Identification of Bacteria by Fatty Acid Profiling with Direct Analysis in Real Time Mass Spectrometry
Authors:
Cody RB, McAlpin CR, Cox CR, Jensen KR, Voorhees KJ.
Goal:
Classify and identify bacteria using DART MS data and multivariate analysis.
Link to Full Text:
https://doi.org/10.1002/rcm.7309
Abstract:
Bacterial fatty acid profiling is a well‐established technique for bacterial identification. Current methods involving esterification and gas chromatography/mass spectrometry (GC/MS) or matrix‐assisted laser desorption/ionization (MALDI) analysis are effective, but there are potential benefits to be gained by investigating ambient ionization methods that can provide rapid analysis without derivatization or additional sample handling. Lipid extracts from colonies of five Gram‐positive and five Gram‐negative pathogenic bacteria were analyzed by Direct Analysis in Real Time (DART) ionization coupled with a time‐of‐flight mass spectrometer. Fatty acid profiles were obtained from the negative‐ion DART mass spectra without additional derivatization or sample preparation. Fatty acid profiles obtained from the deprotonated molecules [M – H]− were found to be highly species‐specific and reproducible. Leave‐one‐out cross validation (LOOCV) for principal component analysis (PCA) showed 100% correct classification accuracy. The results of this preliminary feasibility study show good precision and accuracy, and the fatty acid patterns are clearly distinctive for each of the ten species examined. The speed and ease of analysis and the high classification accuracy for this initial study indicate that DART is an effective method for bacterial fatty acid profiling.
Title:
Comparison of Metal Oxide Catalysts for Pyrolytic MOLI-MS Bacterial Identification
Authors:
Voorhees KJ, Saichek NR, Jensen KR, Harrington PB, Cox CR.
Goal:
Test the effectiveness of several metal oxides for SALDI analysis of bacteria. Bacterial data were analyzed by principal components analysis and cross-validated.
Link to Full Text:
https://doi.org/10.1016/j.jaap.2014.10.016
Abstract:
Six metal oxide catalysts were investigated for effective cleavage and laser ionization of bacterial cell wall phospholipid extracts. Calcium oxide has been utilized in the past for this application, but suffered from degradation of catalytic activity. Following evaluation of the six catalysts, CeO2 was found to be the most stable over time and produced fatty acid profiles of bacterial pathogens similar to freshly activated CaO. A suite of ten bacteria previously studied using CaO catalysis provided cross validation results of analysis of 97% correct correlation for negative-ion data. Cerium oxide catalysis studies yielded cross validation results of 100%. Using analysis of variance–principal component analysis (ANOVA–PCA), four sample sets collected with stored catalyst at 0, 8, 24, and 504 h showed no effect based on long-term catalyst degradation. Supervised learning by a fuzzy rule-building expert system (FuRES) that was validated with training and prediction set partitions independent of CeO2 age and unsupervised data analysis using a dendrogram of Euclidean distance confirmed that the CeO2 catalyst age had no effect on the fatty acid mass spectral profiles.
Title:
Modified MALDI MS Fatty Acid Profiling for Bacterial Identification
Authors:
Voorhees KJ, Jensen KR, McAlpin CR, Rees JC, Cody R, Ubukata M, Cox CR.
Goal:
To classify and identify bacteria using MOLI MS data and multivariate statistical analysis.
Link to Full Text:
https://doi.org/10.1002/jms.3215
Abstract:
Bacterial fatty acid profiling is a well‐established technique for bacterial identification. Ten bacteria were analyzed using both positive‐ and negative‐ion modes with a modified matrix‐assisted laser desorption ionization mass spectrometry (MALDI MS) approach using CaO as a matrix replacement (metal oxide laser ionization MS (MOLI MS)). The results show that reproducible lipid cleavage similar to thermal in situ tetramethyl ammonium hydroxide saponification/derivatization had occurred. Principal component analysis showed that replicates from each organism grouped in a unique space. Cross validation (CV) of spectra from both ionization modes resulted in greater than 94% validation of the data. When CV results were compared for the two ionization modes, negative‐ion data produced a superior outcome. MOLI MS provides clinicians a rapid, reproducible and cost‐effective bacterial diagnostic tool.
Title:
Rapid Detection of Bacillus anthracis by γ Phage Amplification and Lateral Flow Immunochromatography.
Authors:
Cox CR, Jensen KR, Mondesire RR, Voorhees KJ.
Goal:
Use a bacteriophage (a virus that attacks bacteria) in a device similar to the home pregnency test to test for Bacillus anthracis by detecting the bacteriophage amplification.
Link to Full Text:
https://doi.org/10.1016/j.mimet.2015.08.011
Abstract:
New, rapid point-of-need diagnostic methods for Bacillus anthracis detection can enhance civil and military responses to accidental or deliberate dispersal of anthrax as a biological weapon. Current laboratory-based methods for clinical identification of B. anthracis require 12 to 120hrs, and are confirmed by plaque assay using the well-characterized γ typing phage, which requires an additional minimum of 24hrs for bacterial culture. To reduce testing time, the natural specificity of γ phage amplification was investigated in combination with lateral flow immunochromatography (LFI) for rapid, point-of-need B. anthracis detection. Phage-based LFI detection of B. anthracis Sterne was validated over a range of bacterial and phage concentrations with optimal detection achieved in as little as 2hrs from the onset of amplification with a threshold sensitivity of 2.5 x 104cfu/mL. The novel use of γ phage amplification detected with a simple, inexpensive LFI assay provides a rapid, sensitive, highly accurate, and field-deployable method for diagnostic ID of B. anthracis in a fraction of the time required by conventional techniques, and without the need for extensive laboratory culture.
Title:
Improved Quantitative Dynamic Range of Time-of-Flight Mass Spectrometry by Simultaneously Waveform-Averaging and Ion-Counting Data Acquisition
Authors:
Kawai Y, Hondo T, Jensen KR, Toyoda M, Terada K.
Goal:
We combined two techniques for analyzing the output signal in time-of-flight mass spectrometry to measure two analytes with a high concentration difference. We compared the result to a conventional technique.
Link to Full Text:
https://doi.org/10.1007/s13361-018-1967-1
Abstract:
Two different types of data acquisition methods, “averaging mode” and “ion-counting mode”, have been used in a time-of-flight (TOF) mass spectrometry. The most common method is an averaging mode that sums waveform signals obtained from each flight cycle. While it is possible to process many ions arriving at the same TOF in one flight cycle, low-abundance ions are difficult to measure because ion signals are overwhelmed by noises from the detection system. An ion-counting mode is suitable for the detection of such low-concentration ions, but counting loss occurs when two or more ions arrive at the detector within the dead time of the acquisition system. In this study, we introduce a technique that combines two methods to measure target ions with a high concentration difference, i.e., averaging mode and ion-counting mode are used simultaneously for high abundant and trace ions, respectively. By processing waveforms concurrently during data acquisition, one can choose to analyze either or both types of data to achieve a highly quantitative mass spectrum over a wide range of sample concentrations. The result of the argon isotope analysis shows that this method provides a more accurate determination of the isotope ratio compared to averaging mode alone at one-twentieth of the analysis time required by ion-counting alone.
Title:
A New Approach for Accurate Mass Assignment on a Multi-Turn Time-Of-Flight Mass Spectrometer
Authors:
Hondo T, Jensen KR, Aoki J, Toyoda M.
Goal:
We developed a new calibration technique for time-of-flight mass spectrometry using the unique design of the MULUM, which has a infinite, closed orbit flight path.
Link to Full Text:
https://doi.org/10.1177/1469066717723755
Abstract:
A simple, effective accurate mass assignment procedure for a time-of-flight mass spectrometer is desirable. External mass calibration using a mass calibration standard together with an internal mass reference (lock mass) is a common technique for mass assignment, however, using polynomial fitting can result in mass-dependent errors. By using the multi-turn time-of-flight mass spectrometer infiTOF-UHV, we were able to obtain multiple time-of-flight data from an ion monitored under several different numbers of laps that was then used to calculate a mass calibration equation. We have developed a data acquisition system that simultaneously monitors spectra at several different lap conditions with on-the-fly centroid determination and scan law estimation, which is a function of acceleration voltage, flight path, and instrumental time delay. Less than 0.9 mDa mass errors were observed for assigned mass to charge ratios (m/z) ranging between 4 and 134 using only 40Ar+ as a reference. It was also observed that estimating the scan law on-the-fly provides excellent mass drift compensation.
Title:
Instrumentation and Method Development for On-Site Analysis of Helium Isotopes
Authors:
Jensen KR, Hondo T, Sumino H, Toyoda M.
Goal:
We developed a novel method for analyzing helium isotopes that could be employed as an on-site method for measuring volcanic activity.
Link to Full Text:
https://doi.org/10.1021/acs.analchem.7b01299
Abstract:
Helium isotope determination may be useful in measuring volcanic activity and issuing earlier warnings of possible eruptions. A method is presented for measuring the ³He/⁴He ratio in a gas sample using the multi-turn time-of-flight mass spectrometer infiTOF-UHV (MSI-Tokyo Inc., Tokyo Japan) (infiTOF). In contrast to conventional waveform averaging, peaks are determined by counting ion pulses from each time-of-flight trigger. Samples were also measured by conventional magnetic-sector mass spectrometry for comparison. Magnetic sector results were used to designate a standard for infiTOF measurement and to calculate a ratio for each sample measured by infiTOF. Mass assignment error for ultra-pure ³He⁺ standard was 4.30 x 10⁻⁵ Da. Mass assignment error of ⁴He²⁺ and ³He⁺ for sample cylinders was 3.00 x 10⁻⁸ Da and 2.25 x 10⁻⁴ Da, respectively. Abundance ratios determined by infiTOF were found to be within 2% of the abundance ratios determined by magnetic-sector mass spectrometry. Mass drift was less than 50 x 10⁻⁶ Da over ten hours. Sample flow rate was not found to affect the results as long as the reference sample was analyzed under the same conditions. Results indicate that the infiTOF system may be a viable tool for measuring helium isotopes, which may eventually lead to earlier warnings of volcanic activity.
Title:
Analytical Applications of Electron-Monochromator Mass Spectrometry
Authors:
Jensen KR, Voorhees KJ.
Goal:
Provide a comprehensive review of analytical applications the EM-MS system to date.
Link to Full Text:
https://doi.org/10.1002/mas.21395
Abstract:
An electron monochromator (EM) produces an electron beam with a narrow energy distribution that can be utilized with mass spectrometry (MS). The history and development of the EM from an initial research design to a commercial model are reviewed along with MS research applications. An EM incorporated with a mass spectrometer showed significant improvement in sensitivity over traditional methods for negative-ion generation and selectivity for compounds with electrophilic character. Sensitivity of EM-MS has been shown to be 25 fg for hexachlorobenzene in positive-ion mode and 10 fg for nitrobenzene in negative-ion mode. Reports regarding the analysis of chlorinated compounds, explosives, pesticides, phthalates, polychlorodibenzo-p-dioxins, polycyclic aromatic hydrocarbons (PAHs), nitro-polycyclic aromatic hydrocarbons (NPAHs), antioxidants, and bacterial biomarkers are discussed. Additionally, theoretical methods to predict electron-capture properties are presented.
Title:
Effect of biodiesel fuel on “real-world”, nonroad heavy duty diesel engine particulate matter emissions, composition and cytotoxicity.
Authors:
Martin N, Lombard M, Jensen KR, Kelley P, Pratt T, Traviss N.
Goal:
Measure the composition and particulate matter of exhaust from heavy loaders at a recycling center.
Link to Full Text:
https://doi.org/10.1016/j.scitotenv.2016.12.041
Abstract:
Biodiesel is regarded by many as a “greener” alternative fuel to petroleum diesel with potentially lower health risk. However, recent studies examining biodiesel particulate matter (PM) characteristics and health effects are contradictive, and typically utilize PM generated by passenger car engines in laboratory settings. There is a critical need to analyze diesel and biodiesel PM generated in a “real-world” setting where heavy duty-diesel (HDD) engines and commercially purchased fuel are utilized. This study compares the mass concentrations, chemical composition and cytotoxicity of real-world PM from combustion of both petroleum diesel and a waste grease 20% biodiesel blend (B20) at a community recycling center operating HDD nonroad equipment. PM was analyzed for metals, elemental/organic carbon (EC/OC), polycyclic aromatic hydrocarbons (PAHs), and nitro-polycyclic aromatic hydrocarbons (N-PAHs). Cytotoxicity in a human lung epithelial cell line (BEAS-2B) following 24h exposure to the real-world particles was also evaluated. On average, higher concentrations for both EC and OC were measured in diesel PM. B20 PM contained significantly higher levels of Cu and Mo whereas diesel PM contained significantly higher concentrations of Pb. Principal component analysis determined Mo, Cu, and Ni were the metals with the greatest loading factor, suggesting a unique pattern related to the B20 fuel source. Total PAH concentration during diesel fuel use was 1.9 times higher than during B20 operations; however, total N-PAH concentration was 3.3 times higher during B20 use. Diesel PM cytotoxicity was 8.5 times higher than B20 PM (p<0.05) in a BEAS-2B cell line. This study contributes novel data on real-world, nonroad engine sources of metals, PAH and N-PAH species, comparing tailpipe PM vs. PM collected inside the equipment cabin. Results suggest PM generated from burning petroleum diesel in nonroad engines may be more harmful to human health, but the links between exposure, composition and toxicity are not straightforward.
Title:
Metabolomic Analysis of Gingival Crevicular Fluid Using Gas Chromatography/Mass Spectrometry.
Authors:
Ozeki M, Nozaki T, Aoki J, Bamba T, Jensen KR, Murakami S, Toyoda M.
Goal:
To develop a technique for on-site analysis (at a dentist’s office) of gingival crevicular fluid and to identify metabolites indicative of periodontitis.
Link to Full Text:
https://doi.org/10.5702/massspectrometry.A0047
Abstract:
Periodontitis is one of the most prevalent threats to oral health as the most common cause of tooth loss. In order to perform effective treatment, a clinical test that detect sites where disease activity is high and predicts periodontal tissue destruction is strongly desired, however, it is still difficult to prognose the periodontal tissue breakdown on the basis of conventional methods. The aim of this study is to examine the usefulness of gas chromatography/mass spectrometry (GC/MS), which could eventually be used for on-site analysis of metabolites in gingival crevicular fluid (GCF) in order to objectively diagnose periodontitis at a molecular level. GCF samples were collected from two diseased sites (one site with a moderate pocket and another site with a deep pocket) from each patient and from clinically healthy sites of volunteers.Nineteen metabolites were identified using GC/MS. Total ion current chromatograms showed broad differences in metabolite peak patterns between GCF samples obtained from healthy sites, moderate-pocket sites, and deep-pocket sites. The intensity difference of some metabolites was significant at sites with deep pockets compared to healthy sites. Additionally, metabolite intensities at moderate-pocket sites showed an intermediate profile between the severely diseased sites and healthy sites, which suggested that periodontitis progression could be observed with a changing metabolite profile. Principal component analysis confirmed these observations by clearly delineating healthy sites and sites with deep pockets. These results suggest that metabolomic analysis of GCF could be useful for prediction and diagnosis of periodontal disease in a single visit from a patient and provides the groundwork for establishing a new, on-site diagnostic method for periodontitis.
Title:
Formation of 2,6-Di-tert-butyl-4-nitrophenol during Combustion of Diesel Fuel Antioxidant Precursors.
Authors:
Jensen KR, Voorhees KJ, Dempsey EA, Burton J, Ratcliff MA, McCormick RL.
Goal:
Determine if an additive in biodeisel turns into a carcinogenic compound during combustion.
Link to Full Text:
http://dx.doi.org/10.1021/ef501043s
Abstract:
Alkylphenolic antioxidants are commonly used as additives to stabilize gasoline, jet, and diesel fuels. The use of antioxidants in diesel, in particular, has become more common because of increased blending of cracked gas oils and blending of biodiesel. Combustion of these phenolic compounds may have unintended consequences. Nitroaromatic compounds are of particular interest because many are listed as known or suspected carcinogens. During the analysis of previously published data, the identity of an unknown peak was confirmed as 2,6-di-tert-butyl-4-nitrophenol (DBNP), an antioxidant derivative. To determine the source of DBNP, particulate matter samples from combustion of conventional diesel fuels spiked with two possible antioxidant precursors were collected and analyzed by electron monochromator–mass spectrometry. Results show fuel spiked with either 2,6-di-tert-butylphenol or 4-methyl-2,6-di-tert-butylphenol produced higher levels of DBNP than control samples. Because fuels require antioxidant additives to prevent degradation, careful selection of antioxidants is important.
