Browse
Submit Data
Databases
API
Help

Dataset Information

18 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Unlocking Cryptic Metabolites with Mass Spectrometry-Guided Transposon Mutant Selection.

ABSTRACT: The products of most secondary metabolite biosynthetic gene clusters (BGCs) have yet to be discovered, in part due to low expression levels in laboratory cultures. Reporter-guided mutant selection (RGMS) has recently been developed for this purpose: a mutant library is generated and screened, using genetic reporters to a chosen BGC, to select transcriptionally active mutants that then enable the characterization of the "cryptic" metabolite. The requirement for genetic reporters limits the approach to a single pathway within genetically tractable microorganisms. Herein, we utilize untargeted metabolomics in conjunction with transposon mutagenesis to provide a global read-out of secondary metabolism across large numbers of mutants. We employ self-organizing map analytics and imaging mass spectrometry to identify and characterize seven cryptic metabolites from mutant libraries of two different Burkholderia species. Applications of the methodologies reported can expand our understanding of the products and regulation of cryptic BGCs across phylogenetically diverse bacteria.

SUBMITTER: Yoshimura A

PROVIDER: S-EPMC7572862 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

NMR-Guided Mass Spectrometry for Absolute Quantitation of Human Blood Metabolites.

Project description:Broad-based, targeted metabolite profiling using mass spectrometry (MS) has become a major platform used in the field of metabolomics for a variety of applications. However, quantitative MS analysis is challenging owing to numerous factors including (1) the need for, ideally, isotope-labeled internal standards for each metabolite, (2) the fact that such standards may be unavailable or prohibitively costly, (3) the need to maintain the standards' concentrations close to those of the target metabolites, and (4) the alternative use of time-consuming calibration curves for each target metabolite. Here, we introduce a new method in which metabolites from a single serum specimen are quantified on the basis of a recently developed NMR method [ Nagana Gowda et al. Anal. Chem. 2015 , 87 , 706 ] and then used as references for absolute metabolite quantitation using MS. The MS concentrations of 30 metabolites thus derived for test serum samples exhibited excellent correlations with the NMR ones (R2 > 0.99) with a median CV of 3.2%. This NMR-guided-MS quantitation approach is simple and easy to implement and offers new avenues for the routine quantification of blood metabolites using MS. The demonstration that NMR and MS data can be compared and correlated when using identical sample preparations allows improved opportunities to exploit their combined strengths for biomarker discovery and unknown-metabolite identification. Intriguingly, however, metabolites including glutamine, pyroglutamic acid, glucose, and sarcosine correlated poorly with NMR data because of stability issues in their MS analyses or weak or overlapping signals. Such information is potentially important for improving biomarker discovery and biological interpretations. Further, the new quantitation method demonstrated here for human blood serum can in principle be extended to a variety of biological mixtures.

| S-EPMC6260976 | biostudies-literature

Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis.

Project description:Most natural product biosynthetic gene clusters that can be observed bioinformatically are silent. This insight has prompted the development of several methodologies for inducing their expression. One of the more recent methods, termed reporter-guided mutant selection (RGMS), entails creation of a library of mutants that is then screened for the desired phenotype via reporter gene expression. Herein, we apply a similar approach to Burkholderia thailandensis and, using transposon mutagenesis, mutagenize three strains, each carrying a fluorescent reporter in the malleilactone (mal), capistruin (cap), or an unidentified ribosomal peptide (tomm) gene cluster. We show that even a small library of <500 mutants can be used to induce expression of each cluster. We also explore the mechanism of activation and find that inhibition of pyrimidine biosynthesis is linked to the induction of the mal cluster. Both a transposon insertion into pyrF as well as small-molecule-mediated inhibition of PyrF trigger malleilactone biosynthesis. Our results pave the way toward the broad application of RGMS and related approaches to Burkholderia spp.

| S-EPMC7493819 | biostudies-literature

Liquid chromatography quadrupole time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database.

Project description:Untargeted metabolomics provides a comprehensive platform for identifying metabolites whose levels are altered between two or more populations. By using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS), hundreds to thousands of peaks with a unique m/z ratio and retention time are routinely detected from most biological samples in an untargeted profiling experiment. Each peak, termed a metabolomic feature, can be characterized on the basis of its accurate mass, retention time and tandem mass spectral fragmentation pattern. Here a seven-step protocol is suggested for such a characterization by using the METLIN metabolite database. The protocol starts from untargeted metabolomic LC-Q-TOF-MS data that have been analyzed with the bioinformatics program XCMS, and it describes a strategy for selecting interesting features as well as performing subsequent targeted tandem MS. The seven steps described will require 2-4 h to complete per feature, depending on the compound.

| S-EPMC3666335 | biostudies-literature

A cryptic VEGF T-cell epitope: Identification and characterization by mass spectrometry and T-cell assays.

Project description:The vascular endothelial growth factor A (VEGF) is involved in various physiological processes such as angiogenesis or wound healing but is also crucial in pathological events such as tumor growth. Thus, clinical anti-VEGF treatments have been developed which could already prove to have enormous beneficial effects for cancer patients. In this article we describe the first VEGF-derived CD8+ T-cell epitope. The natural HLA ligand SRFGGAVVR was identified by differential mass spectrometry in two primary renal cell carcinomas (RCC) and was significantly over-presented on both tumor tissues. SRFGGAVVR is derived from a cryptic translated region of VEGF presumably by initiation of translation at the non-classical start codon CUG499. SRFGGAVVR specific T-cells were generated in vitro using peptide loaded dendritic cells or artificial antigen presenting cells. They were identified by HLA tetramer analysis after in vitro stimulation. SRFGGAVVR specific CD8+ T-cells were fully functional T-effector cells, which were able to secrete IFN-gamma upon stimulation and killed tumor cells in vitro. Additionally, we have quantitatively analyzed VEGF mRNA and protein levels in RCC tumor and normal tissue samples by gene chip analysis, qRT-PCR, in situ hybridization, and bead based immuno assay. In the future, T-cells directed against VEGF as a tumor associated antigen may represent a possible way of combining peptide-based anti-VEGF immunotherapy with already existent anti-VEGF cancer therapies. Keywords: disease state analysis

2007-06-07 | GSE8050 | GEO

MMP qualitative and quantitative mass spectrometry

Project description:Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media. Mass spectrometry based quantitative proteomics were done on the mixtures to investigate the differences in expression patterns between S40 and S2. Keywords: isotope labeling mass spectrometry, quantitative proteomics

2005-09-20 | GSE2744 | GEO

Mass spectrometry-guided refinement of chemical energy buffers.

Project description:Biocatalytic reactions often require supplying chemical energy and phosphate groups in the form of adenosine triphosphate (ATP). Auxiliary enzymes can be used to convert a reaction by-product-adenosine diphosphate (ADP)-back to ATP. By employing real-time mass spectrometry (RTMS), one can gain an insight into inter-conversions of reactants in multi-enzyme reaction systems and optimize the reaction conditions. In this study, temporal traces of ions corresponding to adenosine monophosphate (AMP), ADP and ATP provided vital information that could be used to adjust activities of the 'buffering enzymes'. Using the RTMS results as a feedback, we also characterized a bienzymatic energy buffer that enables the recovery of ATP in the cases where it is directly hydrolysed to AMP in the main enzymatic reaction. The significance of careful selection of enzyme activities-guided by RTMS-is exemplified in the synthesis of glucose-6-phosphate by hexokinase in the presence of a buffering enzyme, pyruvate kinase. Relative activities of the two enzymes, present in the reaction mixture, influence biosynthetic reaction yields. This observation supports the conclusion that optimization of chemical energy recycling procedures is critical for the biosynthetic reaction economy.

| S-EPMC4950186 | biostudies-literature

Mass Spectrometry Based Molecular 3D-Cartography of Plant Metabolites.

Project description:Plants play an essential part in global carbon fixing through photosynthesis and are the primary food and energy source for humans. Understanding them thoroughly is therefore of highest interest for humanity. Advances in DNA and RNA sequencing and in protein and metabolite analysis allow the systematic description of plant composition at the molecular level. With imaging mass spectrometry, we can now add a spatial level, typically in the micrometer-to-centimeter range, to their compositions, essential for a detailed molecular understanding. Here we present an LC-MS based approach for 3D plant imaging, which is scalable and allows the analysis of entire plants. We applied this approach in a case study to pepper and tomato plants. Together with MS/MS spectra library matching and spectral networking, this non-targeted workflow provides the highest sensitivity and selectivity for the molecular annotations and imaging of plants, laying the foundation for studies of plant metabolism and plant-environment interactions.

| S-EPMC5370242 | biostudies-literature

Profiling Reactive Metabolites via Chemical Trapping and Targeted Mass Spectrometry.

Project description:Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC-MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites.

| S-EPMC4998964 | biostudies-literature

Mass spectrometry combinations for structural characterization of sulfated-steroid metabolites.

Project description:Steroid conjugates, which often occur as metabolites, are challenging to characterize. One application is female-mouse urine, where steroid conjugates serve as important ligands for the pheromone-sensing neurons. Although the two with the highest abundance in mouse urine were previously characterized with mass spectrometry (MS) and NMR to be sulfated steroids, many more exist but remain structurally unresolved. Given that their physical and chemical properties are similar, they are likely to have a sulfated steroid ring structure. Because these compounds occur in trace amounts in mouse urine and elsewhere, their characterization by NMR will be difficult. Thus, MS methods become the primary approach for determining structure. Here, we show that a combination of MS tools is effective for determining the structures of sulfated steroids. Using 4-pregnene analogs, we explored high-resolving power MS (HR-MS) to determine chemical formulae; HD exchange MS (HDX-MS) to determine number of active, exchangeable hydrogens (e.g., OH groups); methoxyamine hydrochloride (MOX) derivatization MS, or reactive desorption electrospray ionization with hydroxylamine to determine the number of carbonyl groups; and tandem MS (MS(n)), high-resolution tandem MS (HRMS/MS), and GC-MS to obtain structural details of the steroid ring. From the fragmentation studies, we deduced three major fragmentation rules for this class of sulfated steroids. We also show that a combined MS approach is effective for determining structure of steroid metabolites, with important implications for targeted metabolomics in general and for the study of mouse social communication in particular.

| S-EPMC4449735 | biostudies-literature

Identifying metabolites by integrating metabolome databases with mass spectrometry cheminformatics.

Project description:Novel metabolites distinct from canonical pathways can be identified through the integration of three cheminformatics tools: BinVestigate, which queries the BinBase gas chromatography-mass spectrometry (GC-MS) metabolome database to match unknowns with biological metadata across over 110,000 samples; MS-DIAL 2.0, a software tool for chromatographic deconvolution of high-resolution GC-MS or liquid chromatography-mass spectrometry (LC-MS); and MS-FINDER 2.0, a structure-elucidation program that uses a combination of 14 metabolome databases in addition to an enzyme promiscuity library. We showcase our workflow by annotating N-methyl-uridine monophosphate (UMP), lysomonogalactosyl-monopalmitin, N-methylalanine, and two propofol derivatives.

| S-EPMC6358022 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data