Publications by authors named "Bao Q Tran"

13 Publications

  • Page 1 of 1

Rapid Denaturing Organic Digestion Method for Targeted Protein Identification and Characterization.

Anal Chem 2021 03 17;93(12):5046-5053. Epub 2021 Mar 17.

Nottingham Trent University, Clifton Campus, College Drive, Nottingham NG11 8NS, U.K.

Bottom-up mass spectrometry-based protein analysis methods employing protease digestion are routinely used to identify and characterize proteins with high specificity and sensitivity. Method performance is generally measured by sequence coverage capability and the total number of characteristic peptides identified, when compared to predicted databases. Limitations to commonly used solvent-based digestion methods currently employed include long digestion times (18-24 h or more), leading to protease autolysis, which also precludes automation, decreases sensitivity, and increases both intra- and inter-day performance variability. This report describes the development and validation of a simple, 5 min tryptic denaturing organic digestion (DOD) method for use with tandem mass spectrometry in bottom-up protein identification and characterization. It has been evaluated across select protein toxins and diagnostic clinical protein targets, substantially improving digestion performance when compared to other solution-based and enzyme-immobilized methods. The method was compared to two currently used bottom-up methods, the 24 h filter-aided sample prep (FASP) and Flash Digest (1 and 4 h) methods. Single proteins used to compare the methods included the ricin light chain, ricin heavy chain, ricin holotoxin, serotype A toxin, enterotoxin B, ribonuclease A, and thyroglobulin. In tests, across the proteins investigated, the 5 min DOD digestion method resulted in sequence coverages ranging from 55 to 100%, with relatively high reproducibility and precision; results were better than or equal to FASP method results and were greatly enhanced when compared to Flash method results. Importantly, DOD method intra- and inter-day precision was much improved as compared to results for both FASP and Flash digestions. These data indicated that the DOD method, when compared to the FASP and Flash Digest methods, dramatically reduced digestion time, while maintaining or improving the ability to detect and characterize targeted proteins, and reduced analytical variability for tryptic digestion, resulting in markedly faster and more precise analyses.
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http://dx.doi.org/10.1021/acs.analchem.0c04143DOI Listing
March 2021

Temporal proteomic profiling reveals changes that support Burkholderia biofilms.

Pathog Dis 2019 03;77(2)

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, MD 21201, USA.

Melioidosis associated with opportunistic pathogen Burkholderia pseudomallei imparts a huge medical burden in Southeast Asia and Australia. At present there is no available human vaccine that protects against B. pseudomallei infection and antibiotic treatments are limited particularly for drug-resistant strains and bacteria in biofilm forms. Biofilm forming bacteria exhibit phenotypic features drastically different to their planktonic states, often exhibiting a diminished response to antimicrobial therapies. Our earlier work on global profiling of bacterial biofilms using transcriptomics and proteomics revealed transcript-decoupled protein abundance in bacterial biofilms. Here we employed reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS) to deduce temporal proteomic differences in planktonic and biofilm forms of Burkholderia thailandensis, which is weakly surrogate model of pathogenic B. pseudomallei as sharing a key element in genomic similarity. The proteomic analysis of B. thailandensis in biofilm versus planktonic states revealed that proteome changes support biofilm survival through decreased abundance of metabolic proteins while increased abundance of stress-related proteins. Interestingly, the protein abundance including for the transcription protein TEX, outer periplasmic TolB protein, and the exopolyphosphatase reveal adaption in bacterial biofilms that facilitate antibiotic tolerance through a non-specific mechanism. The present proteomics study of B. thailandensis biofilms provides a global snapshot of protein abundance differences and antimicrobial sensitivities in planktonic and sessile bacteria.
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http://dx.doi.org/10.1093/femspd/ftz005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482045PMC
March 2019

Proteomic and Metabolomic Profiling Identify Plasma Biomarkers for Exposure to Ultra-low Levels of Carfentanil.

Toxicol Sci 2019 02;167(2):524-535

Biosciences Division, BioDefense Branch, US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 21010, USA.

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.
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http://dx.doi.org/10.1093/toxsci/kfy259DOI Listing
February 2019

Misclassification of blood pressure of Vietnamese adults when only a single measurement is used.

J Am Soc Hypertens 2018 09 30;12(9):671-680. Epub 2018 Jun 30.

Menzies Institute for Medical Research, University of Tasmania, Hobart, Tasmania, Australia.

A single clinic measurement of blood pressure (BP) may be common in low- and middle-income countries because of limited medical resources. This study aimed to examine the potential misclassification error when only one BP measurement is used. Participants (n = 14,706, 53.5% females) aged 25-64 years were selected by multistage stratified cluster sampling from eight provinces, each representing one of the eight geographical regions of Vietnam. Measurements were made using the World Health Organization STEPS protocols. Data were analyzed using complex survey methods. For systolic BP, 62.7% had a higher first reading whereas 30.0% had a lower first reading, and 27.3% had a reduction of at least 5 mmHg whereas 9.6% had an increase of at least 5 mmHg. Irrespective of direction of change, increased variability in BP was associated with greater age, urban living, greater body size and fatness, reduced physical activity levels, elevated glucose, and raised total cholesterol. These measurement variations would lead to substantial misclassification in diagnosis of hypertension based on a single reading because almost 20% of subjects would receive a different diagnosis based on the mean of two readings.
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http://dx.doi.org/10.1016/j.jash.2018.06.015DOI Listing
September 2018

Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery.

Sci Rep 2017 02 21;7:42923. Epub 2017 Feb 21.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA.

Botulinum neurotoxin (BoNT) binds to and internalizes its light chain into presynaptic compartments with exquisite specificity. While the native toxin is extremely lethal, bioengineering of BoNT has the potential to eliminate toxicity without disrupting neuron-specific targeting, thereby creating a molecular vehicle capable of delivering therapeutic cargo into the neuronal cytosol. Building upon previous work, we have developed an atoxic derivative (ad) of BoNT/C1 through rationally designed amino acid substitutions in the metalloprotease domain of wild type (wt) BoNT/C1. To test if BoNT/C1 ad retains neuron-specific targeting without concomitant toxic host responses, we evaluated the localization, activity, and toxicity of BoNT/C1 ad in vitro and in vivo. In neuronal cultures, BoNT/C1 ad light chain is rapidly internalized into presynaptic compartments, but does not cleave SNARE proteins nor impair spontaneous neurotransmitter release. In mice, systemic administration resulted in the specific co-localization of BoNT/C1 ad with diaphragmatic motor nerve terminals. The mouse LD of BoNT/C1 ad is 5 mg/kg, with transient neurological symptoms emerging at sub-lethal doses. Given the low toxicity and highly specific neuron-targeting properties of BoNT/C1 ad, these data suggest that BoNT/C1 ad can be useful as a molecular vehicle for drug delivery to the neuronal cytoplasm.
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http://dx.doi.org/10.1038/srep42923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5318933PMC
February 2017

Global Analysis and Comparison of the Transcriptomes and Proteomes of Group A Biofilms.

mSystems 2016 Nov-Dec;1(6). Epub 2016 Dec 6.

Department of Microbial Pathogenesis, University of Maryland School of Dentistry, Baltimore, Maryland, USA; Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

To gain a better understanding of the genes and proteins involved in group A (GAS; ) biofilm growth, we analyzed the transcriptome, cellular proteome, and cell wall proteome from biofilms at different stages and compared them to those of plankton-stage GAS. Using high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we found distinct expression profiles in the transcriptome and proteome. A total of 46 genes and 41 proteins showed expression across the majority of biofilm time points that was consistently higher or consistently lower than that seen across the majority of planktonic time points. However, there was little overlap between the genes and proteins on these two lists. In line with other studies comparing transcriptomic and proteomic data, the overall correlation between the two data sets was modest. Furthermore, correlation was poorest for biofilm samples. This suggests a high degree of regulation of protein expression by nontranscriptional mechanisms. This report illustrates the benefits and weaknesses of two different approaches to global expression profiling, and it also demonstrates the advantage of using proteomics in conjunction with transcriptomics to gain a more complete picture of global expression within biofilms. In addition, this report provides the fullest characterization of expression patterns in GAS biofilms currently available. Prokaryotes are thought to regulate their proteomes largely at the level of transcription. However, the results from this first set of global transcriptomic and proteomic analyses of paired microbial samples presented here show that this assumption is false for the majority of genes and their products in . In addition, the tenuousness of the link between transcription and translation becomes even more pronounced when microbes exist in a biofilm or a stationary planktonic state. Since the transcriptome level does not usually equal the proteome level, the validity attributed to gene expression studies as well as proteomic studies in microbial analyses must be brought into question. Therefore, the results attained by either approach, whether RNA-seq or shotgun proteomics, must be taken in context and evaluated with particular care since they are by no means interchangeable.
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http://dx.doi.org/10.1128/mSystems.00149-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141267PMC
December 2016

Secreted Effectors Encoded within and outside of the Francisella Pathogenicity Island Promote Intramacrophage Growth.

Cell Host Microbe 2016 Nov;20(5):573-583

Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, School of Medicine, University of Washington, Seattle, WA 98195, USA. Electronic address:

The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens, and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins that we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism.
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http://dx.doi.org/10.1016/j.chom.2016.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384264PMC
November 2016

Human symbionts inject and neutralize antibacterial toxins to persist in the gut.

Proc Natl Acad Sci U S A 2016 Mar 8;113(13):3639-44. Epub 2016 Mar 8.

Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06510; Microbial Sciences Institute, Yale University School of Medicine, West Haven, CT 06516;

The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.
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http://dx.doi.org/10.1073/pnas.1525637113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822603PMC
March 2016

An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.

Cell 2015 Oct 8;163(3):607-19. Epub 2015 Oct 8.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Howard Hughes Medical Institute, Seattle, WA 98195, USA. Electronic address:

Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.
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http://dx.doi.org/10.1016/j.cell.2015.09.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624332PMC
October 2015

Kin cell lysis is a danger signal that activates antibacterial pathways of Pseudomonas aeruginosa.

Elife 2015 Feb 2;4. Epub 2015 Feb 2.

Department of Microbiology, University of Washington, Seattle, United States.

The perception and response to cellular death is an important aspect of multicellular eukaryotic life. For example, damage-associated molecular patterns activate an inflammatory cascade that leads to removal of cellular debris and promotion of healing. We demonstrate that lysis of Pseudomonas aeruginosa cells triggers a program in the remaining population that confers fitness in interspecies co-culture. We find that this program, termed P. aeruginosa response to antagonism (PARA), involves rapid deployment of antibacterial factors and is mediated by the Gac/Rsm global regulatory pathway. Type VI secretion, and, unexpectedly, conjugative type IV secretion within competing bacteria, induce P. aeruginosa lysis and activate PARA, thus providing a mechanism for the enhanced capacity of P. aeruginosa to target bacteria that elaborate these factors. Our finding that bacteria sense damaged kin and respond via a widely distributed pathway to mount a complex response raises the possibility that danger sensing is an evolutionarily conserved process.
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http://dx.doi.org/10.7554/eLife.05701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4348357PMC
February 2015

A type VI secretion-related pathway in Bacteroidetes mediates interbacterial antagonism.

Cell Host Microbe 2014 Aug 25;16(2):227-236. Epub 2014 Jul 25.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA. Electronic address:

Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.
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http://dx.doi.org/10.1016/j.chom.2014.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136423PMC
August 2014

Genetically distinct pathways guide effector export through the type VI secretion system.

Mol Microbiol 2014 May 28;92(3):529-42. Epub 2014 Mar 28.

Department of Microbiology, University of Washington, Seattle, WA, 98195, USA.

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.
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http://dx.doi.org/10.1111/mmi.12571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049467PMC
May 2014

QuaMeter: multivendor performance metrics for LC-MS/MS proteomics instrumentation.

Anal Chem 2012 Jul 27;84(14):5845-50. Epub 2012 Jun 27.

Department of Biomedical Informatics, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8575, United States.

LC-MS/MS-based proteomics studies rely on stable analytical system performance that can be evaluated by objective criteria. The National Institute of Standards and Technology (NIST) introduced the MSQC software to compute diverse metrics from experimental LC-MS/MS data, enabling quality analysis and quality control (QA/QC) of proteomics instrumentation. In practice, however, several attributes of the MSQC software prevent its use for routine instrument monitoring. Here, we present QuaMeter, an open-source tool that improves MSQC in several aspects. QuaMeter can directly read raw data from instruments manufactured by different vendors. The software can work with a wide variety of peptide identification software for improved reliability and flexibility. Finally, QC metrics implemented in QuaMeter are rigorously defined and tested. The source code and binary versions of QuaMeter are available under Apache 2.0 License at http://fenchurch.mc.vanderbilt.edu.
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http://dx.doi.org/10.1021/ac300629pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730131PMC
July 2012