John H. Callahan

Photogenerated Charge Carriers and Reactive Oxygen Species in ZnO/Au Hybrid Nanostructures with Enhanced Photocatalytic and Antibacterial Activity

Semiconductor nanostructures with photocatalytic activity have the potential for many applications including remediation of environmental pollutants and use in antibacterial products. An effective way for promoting photocatalytic activity is depositing noble metal nanoparticles (NPs) on a semiconductor. In this paper, we demonstrated the successful deposition of Au NPs, having sizes smaller than 3 nm, onto ZnO NPs. ZnO/Au hybrid nanostructures having different molar ratios of Au to ZnO were synthesized. It was found that Au nanocomponents even at a very low Au/ZnO molar ratio of 0.2% can greatly enhance the photocatalytic and antibacterial activity of ZnO. Electron spin resonance spectroscopy with spin trapping and spin labeling was used to investigate the enhancing effect of Au NPs on the generation of reactive oxygen species and photoinduced charge carriers. Deposition of Au NPs onto ZnO resulted in a dramatic increase in light-induced generation of hydroxyl radical, superoxide and singlet oxygen, and production of holes and electrons. The enhancing effect of Au was dependent on the molar ratio of Au present in the ZnO/Au nanostructures. Consistent with these results from ESR measurements, ZnO/Au nanostructures also exhibited enhanced photocatalytic and antibacterial activity. These results unveiled the enhanced mechanism of Au on ZnO and these materials have great potential for use in water purification and antibacterial products.

The Chromatographic Role in High Resolution Mass Spectrometry for Non-Targeted Analysis

Resolution improvements in time-of-flight instrumentation and the emergence of the Orbitrap mass spectrometer have researchers using high resolution mass spectrometry to determine elemental compositions and performing screening methods based on the full-scan data from these instruments. This work is focused on examining instrument performance of both a QTOF and a bench-top Orbitrap. In this study, the impact of chromatographic resolution on mass measurement accuracy, mass measurement precision, and ion suppression is examined at a fundamental level. This work was extended to a mixture of over 200 pesticides to determine how well two different software algorithms componentized and correctly identified these compounds under different sets of chromatographic conditions, where co-elution was expected to vary markedly.

Mass Accuracy and Isotopic Abundance Measurements for HR-MS Instrumentation: Capabilities for Non-Targeted Analyses

AbstractThe development of automated non-targeted workflows for small molecule analyses is highly desirable in many areas of research and diagnostics. Sufficient mass and chromatographic resolution is necessary for the detectability of compounds and subsequent componentization and interpretation of ions. The mass accuracy and relative isotopic abundance are critical in correct molecular formulae generation for unknown compounds. While high-resolution instrumentation provides accurate mass information, sample complexity can greatly influence data quality and the measurement of compounds of interest. Two high-resolution instruments, an Orbitrap and a Q-TOF, were evaluated for mass accuracy and relative isotopic abundance with various concentrations of a standard mixture in four complex sample matrices. The overall average ± standard deviation of the mass accuracy was 1.06 ± 0.76xa0ppm and 1.62 ± 1.88xa0ppm for the Orbitrap and the Q-TOF, respectively; however, individual measurements were ± 5xa0ppm for the Orbitrap and greater than 10xa0ppm for the Q-TOF. Relative isotopic abundance measurements for A + 1 were within 5% of the theoretical value if the intensity of the monoisotopic peak was greater than 1E7 for the Orbitrap and 1E5 for the Q-TOF, where an increase in error is observed with a decrease in intensity. Furthermore, complicating factors were found in the data that would impact automated data analysis strategies, including coeluting species that interfere with detectability and relative isotopic abundance measurements. The implications of these findings will be discussed with an emphasis on reasonable expectations from these instruments, guidelines for experimental workflows, data analysis considerations, and software design for non-targeted analyses.n Figureᅟ

Cucurbitane-type triterpenoids from Momordica charantia.

One new cucurbitane-type triterpenoid glycoside, momordicoside U (1), together with five known cucurbitane-type triterpenoids and related glycosides, 3β,7 β,25-trihydroxycucurbita-5,23 (E)-dien-19-al (2), momordicine I (3), momordicine II (4), 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O-β-glucopyranoside (5), and kuguaglycoside G (6), were isolated from the whole plant of Momordica charantia. Their structures were determined by chemical and spectroscopic methods. Momordicoside U (1) was evaluated for insulin secretion activity in an in vitro insulin secretion assay and displayed moderate activity.

Mapping DNA adducts of mitomycin C and decarbamoyl mitomycin C in cell lines using liquid chromatography/electrospray tandem mass spectrometry

The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a nontoxic reductive metabolite of MC formed in cells in situ. Several methods have been used for the analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry, is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia-A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or 10-decarbamoyl mitomycin C (DMC) treatment of the cells, digested to nucleosides, and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored (multiple reaction monitoring). Identification and quantitative analysis were based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 pmol. Three initial applications of this method are reported as follows: (i) differential kinetics of adduct repair in EMT6 cells, (ii) analysis of adducts in MC- or DMC-treated Fanconi Anemia cells, and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: Repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts, and MCF-7 cells as well as EMT6 cells; and DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC.

Evaluation of protein expression in bovine bronchoalveolar fluid following challenge with Mannheimia haemolytica

Proteomics analysis of bovine bronchoalveolar fluid (BAF) following induction of pneumonia with Mannheimia haemolytica using nanoflow liquid chromatography coupled with tandem mass spectrometry (nanoLC‐MS/MS) resulted in the identification of 88 unique proteins. Proteins detected in BAF included antimicrobial peptides (AMPs), complement factors, acute‐phase proteins, protease inhibitors, and proteins involved in oxidation–reduction. Notwithstanding biological variation, differences in relative protein abundance, determined using normalized peptide counts, were detected for select proteins in BAF from genuinely infected versus sham‐infected animals. To demonstrate the applicability of using normalized peptide counts to assess protein expression trends, LC‐MS/MS data for the acute‐phase protein haptoglobin (HPT) were compared with ELISA data, and statistical evaluation of the relationship between the data revealed a strong measure of association. Differences were detected between sham‐ and genuinely infected animals for haptoglobin, as well as the AMPs cathelicidin‐1 and cathelicidin‐4, and inter‐α‐trypsin inhibitor heavy chain‐4, a fairly novel protein involved in the acute phase response. Though the small sample size limited the scope of the inferences, the results indicate the likely importance of AMPs and acute‐phase proteins during respiratory infection, and provide additional information regarding potential mechanisms involved in the bovine mucosal barrier defense.

A discriminant based charge deconvolution analysis pipeline for protein profiling of whole cell extracts using liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry.

A discriminant based charge deconvolution analysis pipeline is proposed. The molecular weight determination (MoWeD) charge deconvolution method was applied directly to the discrimination rules obtained by the fuzzy rule-building expert system (FuRES) pattern classifier. This approach was demonstrated with synthetic electrospray ionization-mass spectra. Identification of the tentative protein biomarkers by bacterial cell extracts of Salmonella enterica serovar typhimurium strains A1 and A19 by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was also demonstrated. The data analysis time was reduced by applying this approach. In addition, this method was less affected by noise and baseline drift.

Combining targeted and nontargeted data analysis for liquid chromatography/high‐resolution mass spectrometric analyses

Increasing importation of food and the diversity of potential contaminants have necessitated more analytical testing of these foods. Historically, mass spectrometric methods for testing foods were confined to monitoring selected ions (SIM or MRM), achieving sensitivity by focusing on targeted ion signals. A limiting factor in this approach is that any contaminants not included on the target list are not typically identified and retrospective data mining is limited. A potential solution is to utilize high-resolution MS to acquire accurate mass full-scan data. Based on the instrumental resolution, these data can be correlated to the actual mass of a contaminant, which would allow for identification of both target compounds and compounds that are not on a target list (nontargets). The focus of this research was to develop software algorithms to provide rapid and accurate data processing of LC/MS data to identify both targeted and nontargeted analytes. Software from a commercial vendor was developed to process LC/MS data and the results were compared to an alternate, vendor-supplied solution. The commercial software performed well and demonstrated the potential for a fully automated processing solution.

Identification and Structural Characterization of Naturally-Occurring Broad-Spectrum Cyclic Antibiotics Isolated from Paenibacillus

AbstractThe rise of antimicrobial resistance necessitates the discovery and/or production of novel antibiotics. Isolated strains of Paenibacillus alvei were previously shown to exhibit antimicrobial activity against a number of pathogens, such as E. coli, Salmonella, and methicillin-resistant Staphylococcus aureus (MRSA). The responsible antimicrobial compounds were isolated from these Paenibacillus strains and a combination of low and high resolution mass spectrometry with multiple-stage tandem mass spectrometry was used for identification. A group of closely related cyclic lipopeptides was identified, differing primarily by fatty acid chain length and one of two possible amino acid substitutions. Variation in the fatty acid length resulted in mass differences of 14 Da and yielded groups of related MSn spectra. Despite the inherent complexity of MS/MS spectra of cyclic compounds, straightforward analysis of these spectra was accomplished by determining differences in complementary product ion series between compounds that differ in molecular weight by 14 Da. The primary peptide sequence assignment was confirmed through genome mining; the combination of these analytical tools represents a workflow that can be used for the identification of complex antibiotics. The compounds also share amino acid sequence similarity to a previously identified broad-spectrum antibiotic isolated from Paenibacillus. The presence of such a wide distribution of related compounds produced by the same organism represents a novel class of broad-spectrum antibiotic compounds.n Graphical Abstractᅟ

Bacterial Identification at the Serovar Level by Top-Down Mass Spectrometry

Food safety efforts require serovar and strain-level specificity of Salmonella contamination to trace back bacterial contamination to its source. Detection methods that require selection of probe-based assays are limited by probe selection. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography-tandem mass spectrometry (LC-MS/MS) facilitates the identification of proteins that result from expressed, serovar-specific non-synonymous single-nucleotide polymorphisms (SNPs). Intact protein expression profiles provide a nontargeted mass spectrometry-based method that facilitates a relatively unbiased snapshot of the expressed proteins in a wide range of bacterial samples and is amenable to both screening and targeted analysis. Such an inherently multiplexed technique facilitates differentiation of closely related bacteria, as well as the detection of un-sequenced or newly acquired SNPs and plasmid proteins that may be specific to a given strain without prior knowledge of the sample. Subsequent identification of expressed proteins, serovar-specific biomarkers, and post-translational modifications by top-down LC-MS/MS is integral to rapid screening development and facilitates collaboration with genome-based methods.