Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Tamarit, Jordi; Hoogh, Anouk de; Òbis, Èlia; Alsina, David; Cabiscol, Elisa; Ros, Joaquim

doi:10.1016/j.jprot.2012.04.046

Cited by 69 publications

(46 citation statements)

References 40 publications

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“…ROS attack proteins, primarily affecting amino acid side chains, and carbonyl group formation is one of the main consequences of this attack. Detection and quantification of these groups has become the most accepted method for measuring oxidative damage to proteins in situations involving oxidative stress (Tamarit and others ). Furthermore, DNA has also been studied as a ROS target, wherein DNA lesions can block genome replication and transcription and, if not remedied, may lead to mutations or aberrations on a large scale, threatening cells viability.…”

Section: Discussionmentioning

confidence: 99%

Protective Role of Flaxseed Oil and Flaxseed Lignan Secoisolariciresinol Diglucoside Against Oxidative Stress in Rats with Metabolic Syndrome

Pilar

Güllich

Oliveira

et al. 2017

Journal of Food Science

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We report that the antioxidant effects attributed to flaxseed are mainly due to its high lignan content, especially that of secoisolariciresinol diglucoside. This is significant because suggests that this compound can be used in isolation to prevent oxidative stress associated with MS. Furthermore, this study was the only one to perform a comparison of the abilities of 2 components of flaxseed to protect against oxidative stress in an MS model, which brings a great advance in the medicine's field, since it indicates another alternative for improve the health and the quality of life of patients with this disorder.

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Section: Discussionmentioning

confidence: 99%

Protective Role of Flaxseed Oil and Flaxseed Lignan Secoisolariciresinol Diglucoside Against Oxidative Stress in Rats with Metabolic Syndrome

Pilar

Güllich

Oliveira

et al. 2017

Journal of Food Science

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“…This method has been widely used in a number of experimental formats such as ELISA, Western blotting and spectrophotometric assays [67–69]. It is also possible to add tags such as biotin and fluorophores using available biotin-hydrazide [70] or BODIPY-hydrazide [71]. Advantages of using biotin or fluorophore tags are that they do not rely on antibody detection, and thus do not suffer from antibody limitations or contaminating immunoglobulins in downstream applications.…”

Section: Detection Of Endogenous Electrophile-protein Adductsmentioning

confidence: 99%

Detection of electrophile-sensitive proteins

Wall

Smith

Ricart

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background Redox signaling is an important emerging mechanism of cellular function. Dysfunctional redox signaling is increasingly implicated in numerous pathologies, including atherosclerosis, diabetes, and cancer. The molecular messengers in this type of signaling are reactive species which can mediate the post-translational modification of specific groups of proteins, thereby effecting functional changes in the modified proteins. Electrophilic compounds comprise one class of reactive species which can participate in redox signaling. Electrophiles modulate cell function via formation of covalent adducts with proteins, particularly cysteine residues. Scope of Review This review will discuss the commonly used methods of detection for electrophile-sensitive proteins, and will highlight the importance of identifying these proteins for studying redox signaling and developing novel therapeutics. Major Conclusions There are several methods which can be used to detect electrophile-sensitive proteins. These include the use of tagged model electrophiles, as well as derivatization of endogenous electrophile-protein adducts. General Significance In order to understand the mechanisms by which electrophiles mediate redox signaling, it is necessary to identify electrophile-sensitive proteins and quantitatively assess adduct formation. Strengths and limitations of these methods will be discussed.

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“…For example, protein carbonylation can directly result from carbonylation of the side chains of amino acids, such as proline, arginine, lysine and threonine [13,14], which may in turn cross-link intracellular proteins. Indirect means of protein modification include addition of oxidized products of carbohydrates or lipids [15,16]. Lipid peroxidation can result in aldehyde-containing products such as 4-hydroxynonenal [17], malondialdehyde, acrolein and heptanal [18].…”

Section: Introductionmentioning

confidence: 99%

“…Popular hydrazine/hydrazide methods use reagents, such as 2, 4-dinitrophenylhydrazine (DNPH) [22,23,24,25,26], biotin-linked hydrazides [27] or fluorophore-hydrazides [16,28]. The resulting hydrazone can then be detected by fluorescence [25,28] or by analytical techniques such as enzyme-linked immunosorbent assay [23,29], Western blot [24], or mass spectrometry [27].…”

Section: Introductionmentioning

confidence: 99%

Detection of oxidative stress-induced carbonylation in live mammalian cells

Mukherjee

Chio

Sackett

et al. 2015

Free Radical Biology and Medicine

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Oxidative stress is often associated with etiology and/or progression of disease conditions, such as cancer, neurodegenerative diseases, and diabetes. At the cellular level, oxidative stress induces carbonylation of biomolecules such as lipids, proteins and DNA. The presence of carbonylcontaining biomolecules as a hallmark of these diseases provides a suitable target for diagnostic detection. Here, a simple, robust method for detecting cellular aldehydes and ketones in live cells using a fluorophore is presented. A hydrazine-functionalized synthetic fluorophore serves as an efficient nucleophile that rapidly reacts with reactive carbonyls in the cellular milieu. The product thus formed exhibits a wavelength shift in the emission maximum accompanied by an increase in emission intensity. The photochemical characteristics of the fluorophore enable the identification of the fluorophore-conjugated cellular biomolecules in the presence of unreacted dye, eliminating the need for removal of excess fluorophore. Moreover, this fluorophore is found to be non-toxic and is thus appropriate for live cell analysis. Utility of the probe is demonstrated in two cell lines, PC3 and A549. Carbonylation resulting from serum starvation and hydrogen peroxide induced stress is detected in both cell lines using fluorescence microscopy and a fluorescence plate reader. The fluorescent signal originates from carbonylated proteins and lipids but not from oxidized DNA, and the majority of the fluorescence signal (>60%) is attributed to fluorophore-conjugated lipid oxidation products. This method should be useful for detecting cellular carbonylation in a high content assay or high throughput assay format.

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Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Cited by 69 publications

References 40 publications

Protective Role of Flaxseed Oil and Flaxseed Lignan Secoisolariciresinol Diglucoside Against Oxidative Stress in Rats with Metabolic Syndrome

Protective Role of Flaxseed Oil and Flaxseed Lignan Secoisolariciresinol Diglucoside Against Oxidative Stress in Rats with Metabolic Syndrome

Detection of electrophile-sensitive proteins

Detection of oxidative stress-induced carbonylation in live mammalian cells

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