Search

Although classical molecular biology assays can provide a measure of cellular response to chemical challenges, they rely on a single biological phenomenon to infer a broader measure of cellular metabolic response. These methods do not always afford the necessary sensitivity to answer questions of subcytotoxic effects, nor do they work for all cell types. Likewise, boutique assays such as cardiomyocyte beat rate may indirectly measure cellular metabolic response, but they too, are limited to measuring a specific biological phenomenon and are often limited to a single cell type. For these reasons, toxicological researchers need new approaches to determine metabolic changes across various doses in differing cell types, especially within the low-dose regime. The data collected herein demonstrate that LC-MS/MS-based untargeted metabolomics with a feature-agnostic view of the data, combined with a suite of statistical methods including an adapted environmental threshold analysis, provides a versatile, robust, and holistic approach to directly monitoring the overall cellular metabolomic response to pesticides. When employing this method in investigating two different cell types, human cardiomyocytes and neurons, this approach revealed separate subcytotoxic metabolomic responses at doses of 0.1 and 1 µM of chlorpyrifos and carbaryl. These findings suggest that this agnostic approach to untargeted metabolomics can provide a new tool for determining effective dose by metabolomics of chemical challenges, such as pesticides, in a direct measurement of metabolomic response that is not cell type-specific or observable using traditional assays. [ABSTRACT FROM AUTHOR]

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical. [ABSTRACT FROM AUTHOR]

Paper spray ionization has grown to become one of the most successful ambient ionization methods within the past decade. Requiring little to no sample preparation and being remarkably simple to construct, this technique has seen application in a wide number of fields. This review approaches the mechanism of how paper spray works, and seeks to better classify what it is and is not in a rapidly expanding field of ambient techniques. Additionally, many applications of the technique in clinical, forensic, environmental, and reaction monitoring regimes are explored. Finally, perspectives towards the future of how paper spray could be utilized will be expanded upon, including unexplored substrates and possibilities for the 'omics space. Image 1 • Paper spray has become one of the most popular ambient ionization techniques. • Significant progress has been made recently in developing methods and applications. • Herein these methods and applications are explored in a variety of research spaces. • Future efforts will include 'omics applications and further design developments. [ABSTRACT FROM AUTHOR]

Despite the recent epidemic of fentanyl abuse, there are few validated assays capable of rapidly detecting these compounds. In order to improve the ability to detect carfentanil at physiologically relevant concentrations, we developed a systems biology approach to discover host-based markers which are specifically amplified upon exposure in a rabbit model. For this work, two "omics" pipelines utilizing mass spectrometry were developed and leveraged. First, a proteomics pipeline was developed to interrogate the blood plasma for protein-based biomarkers. Due to the incredible dynamic range of the plasma protein content, a multi-dimensional fractionation technique was used to partition and more accurately investigate the circulating plasma proteome. Isobaric tandem mass tags were integrated into the workflow to make quantitative assessments across all animals for an extended time course post-exposure. In addition to the proteomics efforts, blood plasma was also processed through an untargeted metabolomics pipeline. This approach allows for the identification of >800 small molecule features. By processing and analyzing data sets in parallel, we were able to identify a unique fingerprint of protein and metabolite perturbations that manifest following exposure to carfentanil. [ABSTRACT FROM AUTHOR]

Rationale: The analysis of chemical warfare agents (CWAs) from ambient atmosphere presents an analytical challenge due to their ease of degradation and volatility. Herein is described a method for derivatizing CWAs directly onto a paper spray substrate prior to analysis. This derivatization allows for much longer times of analysis without sample degradation and with little to no sample preparation. Methods: Derivatization was performed using 2‐[(dimethylamino)methyl] phenol both in‐vial and directly on paper spray cartridges. Solution studies were carried out over time and samples were analyzed via liquid chromatography/tandem mass spectrometry (LC/MS/MS) operated in positive ion mode. Paper spray substrates impregnated with the derivatizing agent prior to CWA vapor capture were also analyzed over time using a mass spectrometer operated in positive ion mode. Results: Use of 2‐[(dimethylamino)methyl] phenol as a paper spray substrate dopant enables derivatization of G‐series compounds into lower volatility complexes. The reaction occurs in solution and in the vapor phase. This new technique effectively traps and captures G‐series agents for analysis while extending the time for which the compound remains absorbed. The complex is highly suitable for direct analysis via paper spray mass spectrometry. Conclusions: Derivatization of paper spray substrates was shown to greatly increase the time for analysis of CWAs. This technique, combined with the vapor phase capture stage outlined previously, allows for rapid, quantitative CWA detection by paper spray ionization with little or no sample preparation. [ABSTRACT FROM AUTHOR]

Paper spray ionization mass spectrometry offers a rapid alternative platform requiring no sample preparation. Aerosolized chemical warfare agent (CWA) simulants trimethyl phosphate, dimethyl methylphosphonate, and diisopropyl methylphosphonate were captured by passing air through a glass fiber filter disk within a disposable paper spray cartridge. CWA simulants were aerosolized at varying concentrations using an in-house built aerosol chamber. A custom 3D-printed holder was designed and built to facilitate the aerosol capture onto the paper spray cartridges. The air flow through each of the collection devices was maintained equally to ensure the same volume of air sampled across methods. Each approach yielded linear calibration curves with R² values between 0.98-0.99 for each compound and similar limits of detection in terms of disbursed aerosol concentration. While the glass fiber filter disk has a higher capture efficiency (≈40%), the paper spray method produces analogous results even with a lower capture efficiency (≈1%). Improvements were made to include glass fiber filters as the substrate within the paper spray cartridge consumable. Glass fiber filters were then treated with ammonium sulfate to decrease chemical interaction with the simulants. This allowed for improved direct aerosol capture efficiency (>40%). Ultimately, the limits of detection were reduced to levels comparable to current worker population limits of 1 × 10-6 mg/m³. [ABSTRACT FROM AUTHOR]

We present an analysis of ambient benzene, toluene, and xylene isomers in the Eagle Ford shale region of southern Texas. In situ air quality measurements using membrane inlet mobile mass spectrometry revealed ambient benzene and toluene concentrations as high as 1000 and 5000 parts-per-billion, respectively, originating from specific sub-processes on unconventional oil and gas well pad sites. The detection of highly variant contamination events attributable to natural gas flaring units, condensate tanks, compressor units, and hydrogen sulfide scavengers indicates that mechanical inefficiencies, and not necessarily the inherent nature of the extraction process as a whole, result in the release of these compounds into the environment. This awareness of ongoing contamination events contributes to an enhanced knowledge of ambient volatile organic compounds on a regional scale. While these reconnaissance measurements on their own do not fully characterize the fluctuations of ambient BTEX concentrations that likely exist in the atmosphere of the Eagle Ford Shale region, they do suggest that contamination events from unconventional oil and gas development can be monitored, controlled, and reduced. [ABSTRACT FROM AUTHOR]

The ability to detect atmospheric effluent from clandestine methamphetamine manufacture is a useful tool for law enforcement. A membrane inlet mass spectrometer is mounted onto an all-electric drive capable hybrid vehicle that samples the atmosphere while in motion. Attributing a latitude and longitude to each spectrum collected, unique chemical fingerprints from clandestine manufacture are then mapped. This location-based mass spectrum data provides a localization to an area of interest. The synthesis of methamphetamine precursors was performed, and the impurities from such reactions were observed. A mock manufacture was setup, and the impurities were introduced into the atmosphere via heating. The detection of products and impurities using this mobile platform has shown the effectiveness of locating and localizing the manufacture of methamphetamine. [ABSTRACT FROM AUTHOR]

The cover image is based on the Research Article On‐Substrate Derivatization for Highly Volatile G‐Series Chemical Warfare Agent Detection via Paper Spray Mass Spectrometry by Phillip M. Mach et al., https://doi.org/10.1002/rcm.8276. [ABSTRACT FROM AUTHOR]

• Targeted method for quantitating eight analytes of human performance in saliva. • Simple sample preparation procedure allows for nearly 100% analyte recovery. • Rapid liquid chromatography run is ideal for high throughput. • Comparison of baseline shows very little difference due to occupational lifestyle. Saliva is increasingly being targeted for metabolic studies due to its non-invasive collection methods. Tracing levels of certain metabolites within biofluids can provide indications for a myriad of physiological conditions. This study was performed on a panel of eight analytes found in saliva that have shown associations with physiological conditions of human performance, such as stress, inflammation, and circadian rhythm. This dual polarity liquid chromatography tandem mass spectrometric (LC MS/MS) method was developed to accommodate a diverse group of analytes including steroids, alkaloids, and neurotransmitters. Samples collected during field exercises from soldiers were compared to those of civilians and baseline levels of each of these compounds was determined in saliva. Although most analytes showed no significant differences between the two populations, relative cortisol levels were higher for soldiers than for civilians. This developed dual polarity LC MS/MS method can be applied to very diverse groups of salivary analytes simultaneously. [ABSTRACT FROM AUTHOR]

Mass spectrometry based 'omics pairs well with organ-on-a-chip-based investigations, which often have limited cellular material for sampling. However, a common issue with these chip-based platforms is well-to-well or chip-to-chip variability in the proteome and metabolome due to factors such as plate edge effects, cellular asynchronization, effluent flow, and limited cell count. This causes high variability in the quantitative multi-omics analysis of samples, potentially masking true biological changes within the system. Solutions to this have been approached via data processing tools and post-acquisition normalization strategies such as constant median, constant sum, and overall signal normalization. Unfortunately, these methods do not adequately correct for the large variations, resulting in a need for increased biological replicates. The methods in this work utilize a dansylation based assay with a subset of labeled metabolites that allow for pre-acquisition normalization to better correlate the biological perturbations that truly occur in chip-based platforms. BCA protein assays were performed in tandem with a proteomics pipeline to achieve pre-acquisition normalization. The CN Bio PhysioMimix was seeded with primary hepatocytes and challenged with VX after six days of culture, and the metabolome and proteome were analyzed using the described normalization methods. A decreased coefficient of variation percentage is achieved, significant changes are observed through the proteome and metabolome, and better classification of biological replicates acquired because of these strategies. [Display omitted] • Organ-on-a-chips are becoming more prevalent. • Organs-on-a-chip display a wide variation in 'omic analysis. • For metabolmic analysis there is a need for pre-acquisition normalization. • Danyslation normalization works well for normalizing organ-on-a-chip samples. [ABSTRACT FROM AUTHOR]

The recent expansion of natural gas and oil extraction using unconventional oil and gas development (UD) practices such as horizontal drilling and hydraulic fracturing has raised questions about the potential for environmental impacts. Prior research has focused on evaluations of air and water quality in particular regions without explicitly considering temporal variation; thus, little is known about the potential effects of UD activity on the environment over longer periods of time. Here, we present an assessment of private well water quality in an area of increasing UD activity over a period of 13 months. We analyzed samples from 42 private water wells located in three contiguous counties on the Eastern Shelf of the Permian Basin in Texas. This area has experienced a rise in UD activity in the last few years, and we analyzed samples in four separate time points to assess variation in groundwater quality over time as UD activities increased. We monitored general water quality parameters as well as several compounds used in UD activities. We found that some constituents remained stable over time, but others experienced significant variation over the period of study. Notable findings include significant changes in total organic carbon and pH along with ephemeral detections of ethanol, bromide, and dichloromethane after the initial sampling phase. These data provide insight into the potentially transient nature of compounds associated with groundwater contamination in areas experiencing UD activity. [ABSTRACT FROM AUTHOR]

Omics-based measurements enable the study of biomolecules in a high-throughput fashion, leading to the characterization and quantification of biological systems. Multi-omics methods aim to incorporate several omics measurements for a more holistic approach, which is crucial for advancing our understanding of the diversity and redundancy of biological systems. Current multi-omics sample preparation methods have achieved proteomics, lipidomics, and metabolomics from individual samples; however, the bioinformatic tools currently available for interpreting data generated from these omics are limited. Alternately, transcriptomics has a wide arsenal of available bioinformatic tools offering intensive sample characterization but has yet to be incorporated into a unified, multi-omics sample preparation technique. Herein we describe the modified bead-enabled accelerated monophasic multi-omics (mBAMM) method, which incorporates RNA extraction for transcriptomics analysis. mBAMM was shown to enable RNA-seq without compromising the isolation of biomolecules for proteomics, lipidomics, and metabolomics. This methodology greatly improves sample characterization and represents a major innovation toward cohesive insights into biological systems.

By characterizing physiological changes that occur in warfighters during simulated combat, we can start to unravel the key biomolecular components that are linked to physical and cognitive performance. Viable field-based sensors for the warfighter must be rapid and noninvasive. In an effort to facilitate this, we applied a multiomics pipeline to characterize the stress response in the saliva of warfighters to correlate biomolecular changes with overall performance and health. In this study, two different stress models were observed - one of chronic stress and one of acute stress. In both models, significant perturbations in the immune, metabolic, and protein manufacturing/processing systems were observed. However, when differentiating between stress models, specific metabolites associated with the "fight or flight" response and protein folding were seen to be discriminate of the acute stress model.

Burkholderia pseudomallei is an opportunistic pathogen that is responsible for the disease melioidosis in humans and animals. The microbe is a tier 1 select agent because it is highly infectious by the aerosol route, it is inherently resistant to multiple antibiotics, and no licensed vaccine currently exists. Naturally acquired infections result from contact with contaminated soil or water sources in regions of endemicity. There have been few reports investigating the molecular mechanism(s) utilized by B. pseudomallei to survive and persist in ecological niches harboring microbial competitors. Here, we report the isolation of Gram-positive bacteria from multiple environmental sources and show that ∼45% of these isolates are inhibited by B. pseudomallei in head-to-head competition assays. Two competition-deficient B. pseudomallei transposon mutants were identified that contained insertion mutations in the hmqA-G operon. This large biosynthetic gene cluster encodes the enzymes that produce a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). Liquid chromatography and mass spectrometry conducted on filter-sterilized culture supernatants revealed five HMAQs and N -oxide derivatives that were produced by the parental strain but were absent in an isogenic hmqD deletion mutant. The results demonstrate that B. pseudomallei inhibits the growth of environmental Gram-positive bacteria in a contact-independent manner via the production of HMAQs by the hmqA-G operon. IMPORTANCE Burkholderia pseudomallei naturally resides in water, soil, and the rhizosphere and its success as an opportunistic pathogen is dependent on the ability to persist in these harsh habitats long enough to come into contact with a susceptible host. In addition to adapting to limiting nutrients and diverse chemical and physical challenges, B. pseudomallei also has to interact with a variety of microbial competitors. Our research shows that one of the ways in which B. pseudomallei competes with Gram-positive environmental bacteria is by exporting a diverse array of closely related antimicrobial secondary metabolites.

Paper spray mass spectrometry has been shown to successfully analyze chemical warfare agent (CWA) simulants. However, due to the volatility differences between the simulants and real G-series (i.e., sarin, soman) CWAs, analysis from an untreated paper substrate proved difficult. To extend the analytical lifetime of these G-agents, metal-organic frameworks (MOFs) were successfully integrated onto the paper spray substrates to increase adsorption and desorption. In this study, several MOFs and nanoparticles were tested to extend the analytical lifetimes of sarin, soman, and cyclosarin on paper spray substrates. It was found that the addition of either UiO-66 or HKUST-1 to the paper substrate increased the analytical lifetime of the G-agents from less than 5 min detectability to at least 50 min.

As wearable fitness devices have gained commercial acceptance, interest in real-time monitoring of an individual's physiological status using noninvasive techniques has grown. Microneedles have been proposed as a minimally invasive technique for sampling the dermal interstitial fluid (ISF) for clinical monitoring and diagnosis, but little is known about its composition. In this study, a novel microneedle array was used to collect dermal ISF from three healthy human donors and compared with matching serum and plasma samples. Using a shotgun quantitative proteomic approach, 407 proteins were quantified with at least one unique peptide, and of those, 135 proteins were differently expressed at least 2-fold. Collectively, these proteins tended to originate from the cytoplasm, membrane bound vesicles, and extracellular vesicular exosomes. Proteomic analysis confirmed previously published work that indicates that ISF is highly similar to both plasma and serum. In this study, less than one percent of proteins were uniquely identified in ISF. Taken together, ISF could serve as a minimally invasive alternative for blood-derived fluids with potential for real-time monitoring applications.