13 research outputs found
Bio-Solid-Phase Extraction/Tandem Mass Spectrometry for Identification of Bioactive Compounds in Mixtures
We
describe a two-step column-based bioassay method with tandem
mass spectrometric detection for rapid identification of bioactive
species in mixtures. The first step uses an immobilized enzyme reactor
(IMER) column interfaced to an electrospray ionization mass spectrometer
(ESI-MS) to identify mixtures containing bioactive compounds (i.e.,
enzyme inhibitors), while the second step uses bioselective solid-phase
extraction (bioSPE) columns to isolate compounds from “hit”
mixtures, which are then identified online by data-dependent ESI-MS.
IMER columns were prepared by entrapment of adenosine deaminase (ADA)
into sol–gel derived monolithic silica columns, and used to
perform a primary IMER screen of mixtures prepared from a bioactive
library, which resulted in four apparent hit compounds. Such columns
did not provide sufficient binding site density to allow bioSPE, and
thus a new column format was developed using ADA that was covalently
immobilized to monolithic silica capillary columns, providing ∼500-fold
more protein binding sites than were present in columns containing
entrapped proteins. Using the covalently linked ADA columns, bioactive
mixtures identified by IMER were infused until a maximum total ion
current was achieved, followed by washing with a buffer to remove
unbound compounds. A harsh wash with 3% acetic acid eluted the strongly
bound ligands and the resulting peak triggered data dependent MS/MS
to identify the ligand, showing that two of the apparent hits were
true ADA inhibitors and demonstrating the ability of this method to
rapidly identify bioactive compounds in mixtures
Differentiation of two human neuroblastoma cell lines alters SV2 expression patterns
Background: The synaptic vesicle glycoprotein 2 (SV2) family is essential to the synaptic machinery involved in neurotransmission and vesicle recycling. The isoforms SV2A, SV2B and SV2C are implicated in neurological diseases such as epilepsy, Alzheimer's and Parkinson's disease. Suitable cell systems for studying regulation of these proteins are essential. Here we present gene expression data of SV2A, SV2B and SV2C in two human neuroblastoma cell lines after differentiation. Methods: Human neuroblastoma cell lines SiMa and IMR-32 were treated for seven days with growth supplements (B-27 and N-2), all-trans-retinoic acid (ATRA) or vasoactive intestinal peptide (VIP) and gene expression levels of SV2 and neuronal targets were analyzed. Results: The two cell lines reacted differently to the treatments, and only one of the three SV2 isoforms was affected at a time. SV2B and choline O-acetyltransferase (CHAT) expression was changed in concert after growth supplement treatment, decreasing in SiMa cells while increasing in IMR-32. ATRA treatment resulted in no detected changes in SV2 expression in either cell line while VIP increased both SV2C and dopamine transporter (DAT) in IMR-32 cells. Conclusion: The synergistic expression patterns between SV2B and CHAT as well as between SV2C and DAT mirror the connectivity between these targets found in disease models and knock-out animals, although here no genetic alteration was made. These cell lines and differentiation treatments could possibly be used to study SV2 regulation and function
Widespread CNS pathology in amyotrophic lateral sclerosis homozygous for the D90A SOD1 mutation
Mutations in the gene encoding the ubiquitously expressed free radical scavenging enzyme superoxide dismutase-1 (SOD1) are found in 2–6% of amyotrophic lateral sclerosis patients. The most frequent SOD1 mutation worldwide is D90A. Amyotrophic lateral sclerosis caused by this mutation has some unusual features: the heredity is usually recessive, the phenotype is stereotypic with slowly evolving motor symptoms beginning in the legs and may also include sensory, autonomic, and urinary bladder involvement. Furthermore, the mutant protein resembles the wild type, with normal content and enzymatic activity in the central nervous system. Here, we report neuropathological findings in nine patients homozygous for the D90A mutation. All nine had numerous small granular inclusions immunoreactive for misfolded SOD1 in motor neurons and glial nuclei in the spinal cord and brainstem. In addition to degeneration of the corticospinal tracts, all patients had degeneration of the dorsal columns. We also found intense gliosis in circumscribed cortical areas of the frontal and temporal lobes and in the insula. In these areas and in adjacent white matter, there were SOD1 staining neuropil threads. A few SOD1-immunopositive cytoplasmic neuronal inclusions were observed in cortical areas, as were glial nuclear inclusions. As suggested by the symptoms and signs and earlier neurophysiological and imaging investigations, the histopathology in patients homozygous for the D90A SOD1 extends beyond the motor system to include cognitive and sensory cortical areas. However, even in the patients that had a symptomatic disease duration of more than 2 or 3 decades and lived into their 70s or 80s, there were no SOD1-inclusion pathology and no typical dysfunction (apart from the musculature) in non-nervous organs. Thus, only specific parts of the CNS seem to be vulnerable to toxicity provoked by homozygously expressed mutant SOD1
Fluorinated Gold Nanoparticles for Nanostructure Imaging Mass Spectrometry
Nanostructure
imaging mass spectrometry (NIMS) with fluorinated
gold nanoparticles (f-AuNPs) is a nanoparticle assisted laser desorption/ionization
approach that requires low laser energy and has demonstrated high
sensitivity. Here we describe NIMS with f-AuNPs for the comprehensive
analysis of metabolites in biological tissues. F-AuNPs assist in desorption/ionization
by laser-induced release of the fluorocarbon chains with minimal background
noise. Since the energy barrier required to release the fluorocarbons
from the AuNPs is minimal, the energy of the laser is maintained in
the low μJ/pulse range, thus limiting metabolite in-source fragmentation.
Electron microscopy analysis of tissue samples after f-AuNP NIMS shows
a distinct “raising” of the surface as compared to matrix
assisted laser desorption ionization ablation, indicative of a gentle
desorption mechanism aiding in the generation of intact molecular
ions. Moreover, the use of perfluorohexane to distribute the f-AuNPs
on the tissue creates a hydrophobic environment minimizing metabolite
solubilization and spatial dislocation. The transfer of the energy
from the incident laser to the analytes through the release of the
fluorocarbon chains similarly enhances the desorption/ionization of
metabolites of different chemical nature, resulting in heterogeneous
metabolome coverage. We performed the approach in a comparative study
of the colon of mice exposed to three different diets. F-AuNP NIMS
allows the direct detection of carbohydrates, lipids, bile acids,
sulfur metabolites, amino acids, nucleotide precursors as well as
other small molecules of varied biological origins. Ultimately, the
diversified molecular coverage obtained provides a broad picture of
a tissue’s metabolic organization
Fluorinated Gold Nanoparticles for Nanostructure Imaging Mass Spectrometry
Nanostructure
imaging mass spectrometry (NIMS) with fluorinated
gold nanoparticles (f-AuNPs) is a nanoparticle assisted laser desorption/ionization
approach that requires low laser energy and has demonstrated high
sensitivity. Here we describe NIMS with f-AuNPs for the comprehensive
analysis of metabolites in biological tissues. F-AuNPs assist in desorption/ionization
by laser-induced release of the fluorocarbon chains with minimal background
noise. Since the energy barrier required to release the fluorocarbons
from the AuNPs is minimal, the energy of the laser is maintained in
the low μJ/pulse range, thus limiting metabolite in-source fragmentation.
Electron microscopy analysis of tissue samples after f-AuNP NIMS shows
a distinct “raising” of the surface as compared to matrix
assisted laser desorption ionization ablation, indicative of a gentle
desorption mechanism aiding in the generation of intact molecular
ions. Moreover, the use of perfluorohexane to distribute the f-AuNPs
on the tissue creates a hydrophobic environment minimizing metabolite
solubilization and spatial dislocation. The transfer of the energy
from the incident laser to the analytes through the release of the
fluorocarbon chains similarly enhances the desorption/ionization of
metabolites of different chemical nature, resulting in heterogeneous
metabolome coverage. We performed the approach in a comparative study
of the colon of mice exposed to three different diets. F-AuNP NIMS
allows the direct detection of carbohydrates, lipids, bile acids,
sulfur metabolites, amino acids, nucleotide precursors as well as
other small molecules of varied biological origins. Ultimately, the
diversified molecular coverage obtained provides a broad picture of
a tissue’s metabolic organization
Systems Biology Guided by XCMS Online Metabolomics
An aim of systems biology is to understand complex interactions between genes, proteins and metabolites by integrating and modeling multiple data sources. We report an 'integrated-omics' approach within XCMS Online1 that automatically superimposes raw metabolomic data onto metabolic pathways and integrates it with transcriptomic and proteomic data (http://XCMSOnline.scripps.edu/)
Global Isotope Metabolomics Reveals Adaptive Strategies for Nitrogen Assimilation
Nitrogen cycling is a microbial metabolic
process essential for
global ecological/agricultural balance. To investigate the link between
the well-established ammonium and the alternative nitrate assimilation
metabolic pathways, global isotope metabolomics was employed to examine
three nitrate reducing bacteria using <sup>15</sup>NO<sub>3</sub> as
a nitrogen source. In contrast to a control (<i>Pseudomonas stutzeri</i> RCH2), the results show that two of the isolates from Oak Ridge,
Tennessee (<i>Pseudomonas</i> N2A2 and N2E2) utilize nitrate
and ammonia for assimilation concurrently with differential labeling
observed across multiple classes of metabolites including amino acids
and nucleotides. The data reveal that the N2A2 and N2E2 strains conserve
nitrogen-containing metabolites, indicating that the nitrate assimilation
pathway is a conservation mechanism for the assimilation of nitrogen.
Co-utilization of nitrate and ammonia is likely an adaption to manage
higher levels of nitrite since the denitrification pathways utilized
by the N2A2 and N2E2 strains from the Oak Ridge site are predisposed
to the accumulation of the toxic nitrite. The use of global isotope
metabolomics allowed for this adaptive strategy to be investigated,
which would otherwise not have been possible to decipher
Systems biology guided by XCMS Online metabolomics.
An aim of systems biology is to understand complex interactions between genes, proteins and metabolites by integrating and modeling multiple data sources. We report an 'integrated-omics' approach within XCMS Online1 that automatically superimposes raw metabolomic data onto metabolic pathways and integrates it with transcriptomic and proteomic data (http://XCMSOnline.scripps.edu/)
Exposome-Scale Investigations Guided by Global Metabolomics, Pathway Analysis, and Cognitive Computing
Concurrent
exposure to a wide variety of xenobiotics and their
combined toxic effects can play a pivotal role in health and disease,
yet are largely unexplored. Investigating the totality of these exposures,
i.e., the “exposome”, and their specific biological
effects constitutes a new paradigm for environmental health but still
lacks high-throughput, user-friendly technology. We demonstrate the
utility of mass spectrometry-based global exposure metabolomics combined
with tailored database queries and cognitive computing for comprehensive
exposure assessment and the straightforward elucidation of biological
effects. The METLIN Exposome database has been redesigned to help
identify environmental toxicants, food contaminants and supplements,
drugs, and antibiotics as well as their biotransformation products,
through its expansion with over 700 000 chemical structures
to now include more than 950 000 unique small molecules. More
importantly, we demonstrate how the XCMS/METLIN platform now allows
for the readout of the biological effect of a toxicant through metabolomic-derived
pathway analysis, and further, artificial intelligence provides a
means of assessing the role of a potential toxicant. The presented
workflow addresses many of the methodological challenges current exposomics
research is facing and will serve to gain a deeper understanding of
the impact of environmental exposures and combinatory toxic effects
on human health