Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia

Izquierdo-Álvarez, Alicia; Ramos, Elena; Villanueva, Joan; Hernansanz-Agustín, Pablo; Fernández‐Rodríguez, Rubén; Tello, Daniel; Carrascal, Montserrat; Martínez‐Ruiz, Antonio

doi:10.1016/j.jprot.2012.06.035

Cited by 37 publications

(37 citation statements)

References 64 publications

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“…6,7,16), this newly developed cysTMTRAQ method is advantageous because it allows researchers to accurately identify and quantify redox cysteines, peptides, and proteins and obtain high efficiency in the correlation between redox changes and total protein changes. Because cysTMT and iTRAQ are employed in the same experiment with the same samples, the sample variation and complication of proteinlevel changes in redox peptide/protein determination are resolved.…”

Section: Discussionmentioning

confidence: 99%

“…Other thiol labeling approaches such as the use of N-ethylmaleimide, biotin-N-[6-(Biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (16), and isotopecoded affinity tags allow specific enrichment of cysteinecontaining peptides, mapping of cysteine modification sites, and duplex experiments in the case of isotope-coded affinity tags (6, 7). To enable multiplexing, 4-or 8-plex iTRAQ tags were recently used to label cysteine-containing peptides isolated from thiol-affinity chromatography (10,11,14,16). Another multiplexing technology, cysTMT, was developed to specifically label cysteines with free thiol groups of proteins from six different samples (9).…”

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confidence: 99%

“…Although these multiplexing technologies have found utility, they do not address the issue of protein turnover in the course of experiments, and many researchers have overlooked this important factor that could lead to misleading results (8,13,14,17). Only a small number of researchers have attempted to compare potential redox changes determined in proteomics experiments to total protein-level changes obtained from parallel or different studies (6,7,16). However, the success of this strategy is often low because many proteins quantified in redox experiments are either absent or not quantified with confidence in total proteomics experiments as a result of experimental variation and mass spectrometry stochastical sampling issues (18,19).…”

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confidence: 99%

“…In addition, the limited number of fluorescent reagents compromises multiplexing capability, and the use of cyanine dyes does not allow mapping of the modified cysteines (6,7). Other thiol labeling approaches such as the use of N-ethylmaleimide, biotin-N-[6-(Biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (16), and isotopecoded affinity tags allow specific enrichment of cysteinecontaining peptides, mapping of cysteine modification sites, and duplex experiments in the case of isotope-coded affinity tags (6,7). To enable multiplexing, 4-or 8-plex iTRAQ tags were recently used to label cysteine-containing peptides isolated from thiol-affinity chromatography (10,11,14,16).…”

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confidence: 99%

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cysTMTRAQ—An Integrative Method for Unbiased Thiol-based Redox Proteomics

Parker

Balmant

Zhu

et al. 2015

Molecular & Cellular Proteomics

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Protein redox regulation plays important roles in many biological processes. Protein cysteine thiols are sensitive to redox changes and may function as redox switches, which turn signaling and metabolic pathways on or off to ensure speedy responses to environmental stimuli or stresses. Here we report a novel integrative proteomics method called cysTMTRAQ that combines two types of isobaric tags, cysteine tandem mass tags and isobaric tag for relative and absolute quantification, in one experiment. The method not only enables simultaneous analysis of cysteine redox changes and total protein level changes, but also allows the determination of bona fide redox modified cysteines in proteins through the correction of protein turnover. Changes in the redox states of protein cysteine thiols serve as regulatory switches in diverse biological processes (1). The redox cycle is regulated by well-known factors such as the ferredoxin-thioredoxin and glutathione-glutaredoxin systems, which reduce oxidized cysteines. Other oxidoreductases and oxidants such as reactive oxygen species act primarily to oxidize cysteine thiol groups (2, 3). In order to map and quantify cysteine redox modifications on the proteome scale, several approaches and methods have been developed, mostly using thiol-specific reagents and isotope tags. Twodimensional gel electrophoresis technology combined with fluorescent dye labeling (e.g. monobromobimane (4, 5) and cyanine dyes (6, 7)) and gel-free technology with isotope tagging (e.g. isotope-coded affinity tagging (6 -8), cysTMT 1 (9), and iTRAQ labeling of enriched cysteine-containing peptides (10 -14)) are often used to identify potential redox-sensitive cysteine residues and quantify redox changes.In addition to the well-known capabilities and limitations associated with two-dimensional gel electrophoresis-based and gel-free approaches (15), each method has its strengths and weaknesses in redox proteomics. For example, the twodimensional gel electrophoresis methods allow the inspection of spot patterns related to redox and protein-level changes. However, spot-volume-based quantification becomes problematic, as each spot often contains more than one protein species from complex samples. In addition, the limited number of fluorescent reagents compromises multiplexing capability, and the use of cyanine dyes does not allow mapping of the modified cysteines (6, 7). Other thiol labeling approaches such as the use of N-ethylmaleimide, biotin-N-[6-(Biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (16), and isotopecoded affinity tags allow specific enrichment of cysteinecontaining peptides, mapping of cysteine modification sites, and duplex experiments in the case of isotope-coded affinity tags (6, 7). To enable multiplexing, 4-or 8-plex iTRAQ tags were recently used to label cysteine-containing peptides isolated from thiol-affinity chromatography (10,11,14,16). Another multiplexing technology, cysTMT, was developed to specifically label cysteines with free thiol groups of proteins from six different sampl...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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cysTMTRAQ—An Integrative Method for Unbiased Thiol-based Redox Proteomics

Parker

Balmant

Zhu

et al. 2015

Molecular & Cellular Proteomics

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“…Comparison of gels stained with these dyes and a total protein stain (e.g. Sypro Ruby) provides exact quantitative and qualitative information about cysteine PTMs in analyzed sample [95]). Some cysteine PTMs can be also detected by specific antibodies.…”

Section: Visualization Of Modified Cysteine Residuesmentioning

confidence: 99%

Advances in purification and separation of posttranslationally modified proteins

Černý

Skalák

Cerná

et al. 2013

Journal of Proteomics

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Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes. The results of enzymatically catalyzed or spontaneous chemical reactions, PTMs form a fourth tier in the gene -transcript -protein cascade, and contribute not only to proteins' biological functions, but also to challenges in their analysis. There have been tremendous advances in proteomics during the last decade. Identification and mapping of PTMs in proteins have improved dramatically, mainly due to constant increases in the sensitivity, speed, accuracy and resolution of mass spectrometry (MS). However, it is also becoming increasingly evident that simple gel-free shotgun MS profiling is unlikely to suffice for comprehensive detection and characterization of proteins and/or protein modifications present in low amounts. Here, we review current approaches for enriching and separating posttranslationally modified proteins, and their MS-independent detection. First, we discuss general approaches for proteome separation, fractionation and enrichment. We then consider the commonest forms of PTMs (phosphorylation, glycosylation and glycation, lipidation, methylation, acetylation, deamidation, ubiquitination and various redox modifications), and the best available methods for detecting and purifying proteins carrying these PTMs.

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Recent advances in proteomics: towards the human proteome

Wang

Huang

Nice

2014

Biomedical Chromatography

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After the successful completion of the Human Genome project in 2003, the next major challenge was to understand when and where the encoded proteins were expressed, and to generate a map of the complex, interconnected pathways, networks and molecular systems (the human proteome) that, taken together, control the workings of all cells, tissues, organs and organisms. Proteomics will be fundamental for such studies. This review summarizes the key discoveries that laid down the foundations for proteomics as we now know it, and describes key recent technological advances that will undoubtedly contribute to achieving the initial goal of the Human Proteome Organization of identifying and characterizing at least one protein product and representative post-translational modifications, single amino acid polymorphisms and splice variant isoforms from the 20,300 human protein-coding genes within the next 10 years. Successful unraveling of the human proteome will undoubtedly improve our understanding of human biology at the cellular level and lay the foundations for improved diagnostic, prognostic, therapeutic and preventive medical outcomes as we enter the era of personalized medicine.

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Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia

Cited by 37 publications

References 64 publications

cysTMTRAQ—An Integrative Method for Unbiased Thiol-based Redox Proteomics

cysTMTRAQ—An Integrative Method for Unbiased Thiol-based Redox Proteomics

Advances in purification and separation of posttranslationally modified proteins

Recent advances in proteomics: towards the human proteome

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