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Unravelling how reactive oxygen species regulate fundamental biological processes is hampered by the lack of an accessible microplate technique to quantify target-specific protein thiol redox state in percentages and moles. To meet this unmet need, we present RedoxiFluor. RedoxiFluor uses two spectrally distinct thiol-reactive fluorescent conjugated reporters, a capture antibody, detector antibody and a standard curve to quantify target-specific protein thiol redox state in relative percentage and molar terms. RedoxiFluor can operate in global mode to assess the redox state of the bulk thiol proteome and can simultaneously assess the redox state of multiple targets in array mode. Extensive proof-of-principle experiments robustly validate the assay principle and the value of each RedoxiFluor mode in diverse biological contexts. In particular, array mode RedoxiFluor shows that the response of redox-regulated phosphatases to lipopolysaccharide (LPS) differs in human monocytes. Specifically, LPS increased PP2A-, SHP1-, PTP1B-, and CD45-specific reversible thiol oxidation without changing the redox state of calcineurin, PTEN, and SHP2. The relative percentage and molar terms are interpretationally useful and define the most complete and extensive microplate redox analysis achieved to date. RedoxiFluor is a new antibody technology with the power to quantify relative target-specific protein thiol redox state in percentages and moles relative to the bulk thiol proteome and selected other targets in a widely accessible, simple and easily implementable microplate format.
Akter,
Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.
2018, Pubmed
Akter,
Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.
2018,
Pubmed
Alcock,
Chemical methods for mapping cysteine oxidation.
2018,
Pubmed
Baty,
Detection of oxidant sensitive thiol proteins by fluorescence labeling and two-dimensional electrophoresis.
2002,
Pubmed
Bazopoulou,
Developmental ROS individualizes organismal stress resistance and lifespan.
2019,
Pubmed
Behring,
Spatial and temporal alterations in protein structure by EGF regulate cryptic cysteine oxidation.
2020,
Pubmed
Bersweiler,
A scaffold protein that chaperones a cysteine-sulfenic acid in H2O2 signaling.
2017,
Pubmed
Bolduc,
Peroxiredoxins wear many hats: Factors that fashion their peroxide sensing personalities.
2021,
Pubmed
Buettner,
Quantitative redox biology: an approach to understand the role of reactive species in defining the cellular redox environment.
2013,
Pubmed
Burgoyne,
The PEG-switch assay: a fast semi-quantitative method to determine protein reversible cysteine oxidation.
2013,
Pubmed
Clift,
Restarting life: fertilization and the transition from meiosis to mitosis.
2013,
Pubmed
Cobley,
Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation.
2019,
Pubmed
,
Xenbase
Cobley,
Proteomic strategies to unravel age-related redox signalling defects in skeletal muscle.
2019,
Pubmed
Cobley,
Immunological Techniques to Assess Protein Thiol Redox State: Opportunities, Challenges and Solutions.
2020,
Pubmed
Cobley,
Lifelong training preserves some redox-regulated adaptive responses after an acute exercise stimulus in aged human skeletal muscle.
2014,
Pubmed
Cobley,
Reversible Thiol Oxidation Inhibits the Mitochondrial ATP Synthase in Xenopus Laevis Oocytes.
2020,
Pubmed
,
Xenbase
Dansen,
Redox-sensitive cysteines bridge p300/CBP-mediated acetylation and FoxO4 activity.
2009,
Pubmed
Day,
Stoichiometric Thiol Redox Proteomics for Quantifying Cellular Responses to Perturbations.
2021,
Pubmed
Forman,
Targeting oxidative stress in disease: promise and limitations of antioxidant therapy.
2021,
Pubmed
Go,
The cysteine proteome.
2015,
Pubmed
Halliwell,
Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean?
2004,
Pubmed
Hansen,
Quantifying the global cellular thiol-disulfide status.
2009,
Pubmed
Hansen,
An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations.
2009,
Pubmed
Held,
Redox Systems Biology: Harnessing the Sentinels of the Cysteine Redoxome.
2020,
Pubmed
Held,
Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach.
2010,
Pubmed
Hochgräfe,
Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis.
2005,
Pubmed
Holmström,
Cellular mechanisms and physiological consequences of redox-dependent signalling.
2014,
Pubmed
Huang,
ROS regulated reversible protein phase separation synchronizes plant flowering.
2021,
Pubmed
Hurd,
Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling.
2007,
Pubmed
Jinek,
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
2012,
Pubmed
Kim,
Efficient site-specific labeling of proteins via cysteines.
2008,
Pubmed
Kobayashi,
Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.
2004,
Pubmed
Leichert,
Quantifying changes in the thiol redox proteome upon oxidative stress in vivo.
2008,
Pubmed
Lennicke,
Redox metabolism: ROS as specific molecular regulators of cell signaling and function.
2021,
Pubmed
Lennicke,
Redox regulation of the insulin signalling pathway.
2021,
Pubmed
Lim,
A reaction-diffusion model of cytosolic hydrogen peroxide.
2016,
Pubmed
Londhe,
Regulation of PTP1B activation through disruption of redox-complex formation.
2020,
Pubmed
Low,
Peroxiredoxin 2 and peroxide metabolism in the erythrocyte.
2008,
Pubmed
Low,
Peroxiredoxin 2 functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte.
2007,
Pubmed
Luo,
Thiol-disulphide independent in-cell trapping for the identification of peroxiredoxin 2 interactors.
2021,
Pubmed
Makmura,
Development of a sensitive assay to detect reversibly oxidized protein cysteine sulfhydryl groups.
2001,
Pubmed
Manford,
A Cellular Mechanism to Detect and Alleviate Reductive Stress.
2020,
Pubmed
Manford,
Structural basis and regulation of the reductive stress response.
2021,
Pubmed
Mason,
Immuno-spin trapping of macromolecules free radicals in vitro and in vivo - One stop shopping for free radical detection.
2019,
Pubmed
Muyldermans,
A guide to: generation and design of nanobodies.
2021,
Pubmed
Noble,
ALISA: A microplate assay to measure protein thiol redox state.
2021,
Pubmed
,
Xenbase
Pak,
Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function.
2020,
Pubmed
Parvez,
Redox Signaling by Reactive Electrophiles and Oxidants.
2018,
Pubmed
Paulsen,
Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery.
2013,
Pubmed
Paulsen,
Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity.
2011,
Pubmed
Pillay,
Quantitative measures for redox signaling.
2016,
Pubmed
Poole,
Introduction to approaches and tools for the evaluation of protein cysteine oxidation.
2020,
Pubmed
Raman,
Redox inhibition of protein phosphatase PP2A: Potential implications in oncogenesis and its progression.
2019,
Pubmed
Shi,
Activity-Based Sensing for Site-Specific Proteomic Analysis of Cysteine Oxidation.
2020,
Pubmed
Sies,
Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
2020,
Pubmed
Sobotta,
Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling.
2015,
Pubmed
Stöcker,
The Conundrum of Hydrogen Peroxide Signaling and the Emerging Role of Peroxiredoxins as Redox Relay Hubs.
2018,
Pubmed
Szczepanowska,
A salvage pathway maintains highly functional respiratory complex I.
2020,
Pubmed
Tyagarajan,
Thiol-reactive dyes for fluorescence labeling of proteomic samples.
2003,
Pubmed
van Leeuwen,
Click-PEGylation - A mobility shift approach to assess the redox state of cysteines in candidate proteins.
2017,
Pubmed
Wellner,
Rapid generation of potent antibodies by autonomous hypermutation in yeast.
2021,
Pubmed
Winterbourn,
Thiol chemistry and specificity in redox signaling.
2008,
Pubmed
Winterbourn,
Reconciling the chemistry and biology of reactive oxygen species.
2008,
Pubmed
Winterbourn,
Doxorubicin-dependent lipid peroxidation at low partial pressures of O2.
1985,
Pubmed
Winther,
Quantification of thiols and disulfides.
2014,
Pubmed
Xiao,
A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging.
2020,
Pubmed
Yang,
The Expanding Landscape of the Thiol Redox Proteome.
2016,
Pubmed
