Trevor Glaros

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses

BackgroundThe bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers.ResultsReasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei.ConclusionsOur results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

Targeted Protein Detection Using an All-in-One Mass Spectrometry Cartridge

We developed a simple 3D printed cartridge for mass spectrometry (MS) targeted detection of plasma proteins, including post-translational modifications (PTMs). The cartridge uses an integrated antibody enrichment column to preconcentrate the protein target as well as a novel built-in substrate to ionize the protein targets for MS detection. We show several examples of using this cartridge to perform rapid detection of clinically significant proteoforms from plasma samples.

Proteomic Characterization of Dermal Interstitial Fluid Extracted Using a Novel Microneedle-Assisted Technique

As wearable fitness devices have gained commercial acceptance, interest in real-time monitoring of an individuals 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.

Metal–Organic Framework Modified Glass Substrate for Analysis of Highly Volatile Chemical Warfare Agents by Paper Spray Mass Spectrometry

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.

Activity Based Protein Profiling Leads to Identification of Novel Protein Targets for Nerve Agent VX

Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions, which could be useful for the development of novel diagnostics or potential adjuvant therapeutics.

Direct Analysis of Aerosolized Chemical Warfare Simulants Captured on a Modified Glass-Based Substrate by “Paper-Spray” Ionization

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.

On-Substrate Derivatization for Highly Volatile G-Series Chemical Warfare Agent Detection via Paper Spray Mass Spectrometry

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.

Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virion and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication

ABSTRACT Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus, to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts, and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro, and Chikungunya viruses. The most significant finding was that in addition to the host proteins, SINV nonstructural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics. IMPORTANCE Pathogenic alphaviruses, such as Chikungunya and Mayaro viruses, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens, such as dengue fever, West Nile, and Yellow fever viruses. With few exceptions, there are no vaccines or prophylactics for these agents, leaving one-third of the world population at risk of infection. Identifying effective antivirals has been a long-term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses.

Extraction and biomolecular analysis of dermal interstitial fluid collected with hollow microneedles

Dermal interstitial fluid (ISF) is an underutilized information-rich biofluid potentially useful in health status monitoring applications whose contents remain challenging to characterize. Here, we present a facile microneedle approach for dermal ISF extraction with minimal pain and no blistering for human subjects and rats. Extracted ISF volumes were sufficient for determining transcriptome, and proteome signatures. We noted similar profiles in ISF, serum, and plasma samples, suggesting that ISF can be a proxy for direct blood sampling. Dynamic changes in RNA-seq were recorded in ISF from induced hypoxia conditions. Finally, we report the first isolation and characterization, to our knowledge, of exosomes from dermal ISF. The ISF exosome concentration is 12–13 times more enriched when compared to plasma and serum and represents a previously unexplored biofluid for exosome isolation. This minimally invasive extraction approach can enable mechanistic studies of ISF and demonstrates the potential of ISF for real-time health monitoring applications.Philip Miller et al. present an approach for extracting dermal interstitial fluid (ISF) using an array of hollow microneedles in a cylindrical substrate that minimizes skin compression and tissue damage. They extract larger volumes of ISF suitable for downstream analyses, compared to previous reports.

Effects of organophosphates on the regulation of mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) are multipotent cells located within various adult tissues. Recent literature has reported that human bone marrow-derived MSCs express active acetylcholinesterase (AChE) and that disruption of AChE activity by organophosphate (OP) chemicals decreases the ability of MSCs to differentiate into osteoblasts. The potential role of AChE in regulating MSC proliferation and differentiation is currently unknown. In the present study, we demonstrate that MSCs exposed to OPs have both decreased AChE activity and abundance. In addition, exposure to these OPs induced cellular death while decreasing cellular proliferation. Exposures to these compounds also reduced the adipogenic/osteogenic differentiation potentials of the MSCs. To elucidate the possible role of AChE in MSCs signaling following OP exposure, we captured potential AChE binding partners by performing polyhistidine (His8)-tagged AChE pulldowns, followed by protein identification using liquid chromatography mass spectrometry (LC-MS). Using this method, we determined that the focal adhesion protein, vinculin, is a potential binding partner with AChE in MSCs and these initial findings were confirmed with follow-up co-immunoprecipitation experiments. Identifying AChE binding partners helps to determine potential pathways associated with MSC proliferation and differentiation, and this understanding could lead to the development of future MSC-based tissue repair therapies.