Browse
Submit Data
Databases
API
Help

Dataset Information

12 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Measurement of H2O2 within living Drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix.

ABSTRACT: Hydrogen peroxide (H(2)O(2)) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H(2)O(2) in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H(2)O(2) levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H(2)O(2) to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H(2)O(2) that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H(2)O(2) with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H(2)O(2) correlates with aging, it may not be causative.

SUBMITTER: Cocheme HM

PROVIDER: S-EPMC4413513 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Ratiometric pulse-chase amidination mass spectrometry as a probe of biomolecular complex formation.

Project description:Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse-chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from Bacillus subtilis as a model system. CsoR in various allosteric states was reacted with S-methyl thioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methyl thiopropionimidate (SMTP) (? = 14 amu), quenched and digested with chymotrypsin or Glu-C protease, and peptides were quantified by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and/or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We show that the reactivities of individual lysines from peptides containing up to three Lys residues are readily quantified using this method. New insights into operator DNA binding and the Cu(I)-mediated structural transition in the tetrameric copper sensor CsoR are also obtained.

| S-EPMC3246313 | biostudies-literature

A New Two-Photon Ratiometric Fluorescent Probe for Detecting Alkaline Phosphatase in Living Cells.

Project description:Alkaline phosphatase (ALP) is an important diagnostic indicator of many human diseases. To quantitatively track ALP in biosystems, herein, for the first time, we report an efficient two-photon ratiometric fluorescent probe, termed probe 1 and based on classic naphthalene derivatives with a donor-π-acceptor (D-π-A) structure and deprotection of the phosphoric acid moiety by ALP. The presence of ALP causes the cleave of the phosphate group from naphthalene derivatives and the phosphate group changes the ability of the intramolecular charge transfer (ICT) and remarkably alters the probe's photophysical properties, thus an obvious ratiometric signal with an isoemissive point is observed. The fluorescence intensity ratio displayed a linear relationship against the concentration of ALP in the concentration range from 20 to 180 U/L with the limit of detection of 2.3 U/L. Additionally, the probe 1 is further used for fluorescence imaging of ALP in living cells under one-photon excitation (405 nm) or two-photon excitation (720 nm), which showed a high resolution imaging, thus demonstrating its practical application in biological systems.

| S-EPMC6273910 | biostudies-literature

Rapid-Response and Highly Sensitive Boronate Derivative-Based Fluorescence Probe for Detecting H2O2 in Living Cells.

Project description:Intracellular H2O2 monitoring is important and has driven researchers to pursue advancements for the rapid identification of H2O2, since H2O2 is short-lived in cell lines. An arylboronate derivative has been investigated as a chemospecific fluorescence recognition agent for H2O2. Triphenylimidazoleoxadiazolephenyl (TPIOP) boronate was contrived as a novel candidate for the rapid and sensitive recognition of H2O2. The probe was conjugated using the TPIOP functional group acting as an excellent fluorescent enhancer. The TPIOP group stimulated the polarization of C-B bond due to its extended ?-conjugation, which included heteroatoms, and induced the production of rapid signal because of the highly polar C-B bond along with the corresponding boronate unit. While H2O2 reacts with TPIOP boronate, its nucleophilic addition to the boron generates a charged tetrahedral boronate complex, and then the C-B bond migrates toward one of the electrophilic peroxide oxygen atoms. The resulting boronate ester is then hydrolyzed by water into a phenol, which significantly enhances fluorescence through aggregation-induced emission. The TPIOP boronate probe responded to H2O2 rapidly, within 2?min, and exhibited high sensitivity with a limit of detection of 8?nM and a 1000-fold selectivity in the presence of other reactive oxygen species. Therefore, the developed TPIOP boronate chemodosimeter was successfully utilized to visualize and quantify intracellular H2O2 from human breast cancer (MCF-7) cells, as well as gaseous and aqueous H2O2 from environmental samples using Whatman paper strips coated with TPIOP boronate.

| S-EPMC6525842 | biostudies-literature

Imaging of fluoride ion in living cells and tissues with a two-photon ratiometric fluorescence probe.

Project description:A reaction-based two-photon (TP) ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS) as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM).

| S-EPMC4327094 | biostudies-literature

Live-Cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe.

Project description:A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of Aspergillus niger. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in Aspergillus. Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin-treated hyphae. Pronounced, longitudinal cytoplasmic pH gradients were not observed in the apical 20 microm of actively growing hyphae at the periphery of 18-h-old colonies. The cytoplasmic pH remained unchanged after prolonged growth in buffered medium with pH values between 2.5 or 9.5. Sudden changes in external pH significantly changed cytoplasmic pH by <1.3 pH units, but it returned to its original value within 20 min following treatment. The weak acid and antifungal food preservative sorbic acid caused prolonged, concentration-dependent intracellular acidification. The inhibition of ATPases with N-ethylmaleimide, dicychlohexylcarbodimide, or sodium azide caused the cytoplasmic pH to decrease by <1 pH unit. Treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone or cyanide p-(trifluoromethoxy) phenylhydrazone reduced the cytoplasmic pH by <1 pH unit. In older hyphae from 32-h-old cultures, RaVC became sequestered within large vacuoles, which were shown to have pH values between 6.2 and 6.5. Overall, our study demonstrates that RaVC is an excellent probe for visualizing and quantifying intracellular pH in living fungal hyphae.

| S-EPMC2681602 | biostudies-literature

An Endoperoxide Reactivity-Based FRET Probe for Ratiometric Fluorescence Imaging of Labile Iron Pools in Living Cells.

Project description:Iron is essential for sustaining life, as its ability to cycle between multiple oxidation states is critical for catalyzing chemical transformations in biological systems. However, without proper regulation, this same redox capacity can trigger oxidative stress events that contribute to aging along with diseases ranging from cancer to cardiovascular and neurodegenerative disorders. Despite its importance, methods for monitoring biological iron bound weakly to cellular ligands-the labile iron pool-to generate a response that preserves spatial and temporal information remain limited, owing to the potent fluorescence quenching ability of iron. We report the design, synthesis, and biological evaluation of FRET Iron Probe 1 (FIP-1), a reactivity-based probe that enables ratiometric fluorescence imaging of labile iron pools in living systems. Inspired by antimalarial natural products and related therapeutics, FIP-1 links two fluorophores (fluorescein and Cy3) through an Fe(II)-cleavable endoperoxide bridge, where Fe(II)-triggered peroxide cleavage leads to a decrease in fluorescence resonance energy transfer (FRET) from the fluorescein donor to Cy3 acceptor by splitting these two dyes into separate fragments. FIP-1 responds to Fe(II) in aqueous buffer with selectivity over competing metal ions and is capable of detecting changes in labile iron pools within living cells with iron supplementation and/or depletion. Moreover, application of FIP-1 to a model of ferroptosis reveals a change in labile iron pools during this form of cell death, providing a starting point to study iron signaling in living systems.

| S-EPMC5749882 | biostudies-literature

An ESIPT-Based Ratiometric Fluorescent Probe for Highly Sensitive and Rapid Detection of Sulfite in Living Cells.

Project description:The novel ratiometric fluorescent probe HPQRB with an ESIPT effect based on Michael addition for highly sensitive and fast detection of sulfite in living HepG2 cells is reported. HPQRB can be easily synthesized by a two-step condensation reaction. HPQRB has a large emission shift (??=116?nm), which is beneficial for fluorescence imaging research, and its sulfite-responsive site is based on a rhodamine-like structure with the emission peak at 566?nm, which decreases with increasing sulfite concentration. and its HPQ structure always has an ESIPT effect throughout the reaction process, keeping the emission peak at 450?nm as a self-reference. In particular, HPQRB has high selectivity for sulfite and responds quickly (within 30?s) with a low detection limit (44?nM). Furthermore, HPQRB has been successfully used for fluorescence imaging of sulfite in HepG2 cells, demonstrating the superior ability to detect sulfite under physiological conditions.

| S-EPMC6779067 | biostudies-literature

Visualizing ascorbate-triggered release of labile copper within living cells using a ratiometric fluorescent sensor.

Project description:We present the synthesis, properties, and biological applications of Ratio-Coppersensor-1 (RCS1), a new water-soluble fluorescent sensor for ratiometric imaging of copper in living cells. RCS1 combines an asymmetric BODIPY reporter and thioether-based ligand receptor to provide high selectivity and sensitivity for Cu(+) over other biologically relevant metal ions, including Cu(2+) and Zn(2+), a ca. 20-fold fluorescence ratio change upon Cu(+) binding, and visible excitation and emission profiles compatible with standard fluorescence microscopy filter sets. Live-cell confocal microscopy experiments show that RCS1 is membrane-permeable and can sense changes in the levels of labile Cu(+) pools within living cells by ratiometric imaging, including expansion of endogenous stores of exchangeable intracellular Cu(+) triggered by ascorbate stimulation in kidney and brain cells.

| S-EPMC2819030 | biostudies-literature

A highly specific ratiometric two-photon fluorescent probe to detect dipeptidyl peptidase IV in plasma and living systems.

Project description:In this study, a highly specific ratiometric two-photon fluorescent probe GP-BAN was developed and well-characterized to monitor dipeptidyl peptidase IV in plasma and living systems. GP-BAN was designed on the basis of the catalytic properties and substrate preference of DPP-IV, and it could be readily hydrolyzed upon addition of DPP-IV under physiological conditions. Both reaction phenotyping and inhibition assays demonstrated that GP-BAN displayed good reactivity and high selectivity towards DPP-IV over other human serine hydrolases including FAP, DPP-VIII, and DPP-IX. The probe was successfully used to monitor the real activities of DPP-IV in complex biological systems including diluted plasma, while it could be used for high throughput screening of DPP-IV inhibitors by using human plasma or tissue preparations as enzyme sources. As a two-photon fluorescent probe, GP-BAN was also successfully used for two-photon imaging of endogenous DPP-IV in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue penetration ability. Taken together, a ratiometric two-photon fluorescent probe GP-BAN was developed and well-characterized for highly selective and sensitive detection of DPP-IV in complex biological systems, which could serve as a promising imaging tool to explore the biological functions and physiological roles of this key enzyme in living systems.

| S-EPMC7127234 | biostudies-literature

Selective detection of endogenous H₂S in living cells and the mouse hippocampus using a ratiometric fluorescent probe.

Project description:As one of three gasotransmitters, the fundamental signalling roles of hydrogen sulphide are receiving increasing attention. New tools for the accurate detection of hydrogen sulphide in cells and tissues are in demand to probe its biological functions. We report the p-nitrobenzyl-based ratiometric fluorescent probe RHP-2, which features a low detection limit, high selectivity and good photostability. The emission intensity ratios had a good linear relationship with the sulphide concentrations in PBS buffer and bovine serum. Our probe was applied to the ratiometric determination and imaging of endogenous H2S in living cells. Furthermore, RHP-2 was used as an effective tool to measure endogenous H2S in the mouse hippocampus. We observed a significant reduction in sulphide concentrations and downregulated expression of cystathionine β-synthetase (CBS) mRNA and CBS protein in the mouse hippocampus in a chronic unpredictable mild stress (CUMS)-induced depression model. These data suggested that decreased concentrations of endogenous H2S may be involved in the pathogenesis of chronic stress depression.

| S-EPMC5376197 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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