Differential protein labeling with thiol‐reactive infrared DY‐680 and DY‐780 maleimides and analysis by two‐dimensional gel electrophoresis

Riederer, Irène M.; Riederer, Beat M.

doi:10.1002/pmic.200601007

Cited by 17 publications

(22 citation statements)

References 8 publications

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“…Proteins (2.5 mg) were solubilized, reduced and desalted before being treated with 200 μg of DY-680 or DY-780 in DMF. While this labeling procedure reduces the initial protein pool during the desalting and concentration steps, DY-680 was shown to be highly sensitive with a LLD of 10 fg (labeled tubulin, with no reported standard deviation) [153]. Once again, however, proteins without accessible cys residues were not detected by these maleimides, indicating a level of inter-protein variability that will be sample dependent.…”

Section: Reactive Fluorescent Dyesmentioning

confidence: 98%

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Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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Section: Reactive Fluorescent Dyesmentioning

confidence: 98%

“…Additional maleimides include the newly developed DY-680 and DY-780 dyes which are infrared thiol-reactive dyes; these were used to compare newborn and adult murine brains [153]. Proteins (2.5 mg) were solubilized, reduced and desalted before being treated with 200 μg of DY-680 or DY-780 in DMF.…”

Section: Reactive Fluorescent Dyesmentioning

confidence: 99%

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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“…DY-maleimide dyes (1 mg dye; 189 Euro) were found to be approximately 50 Â less expensive than CyDye-DIGE saturation-labeling dyes (1380 Euro/kit; 12 multiplexed samples). Nevertheless, if additional 2-DE differential proteomic analyses are needed, CyDye-DIGE minimal labeling or the recently developed infrared maleimide dye-based DIGE method could be applied [15,20,[38][39][40]. In summary, our 2-DE studies suggest that fluorescent DYmaleimide protein labeling is superior to standard fluorescent post-electrophoresis staining, leading to a threefold increase in the number of protein spots detectable, a twofold higher protein spot quality, and an up to 100 times more sensitive pattern intensity.…”

Section: Discussionmentioning

confidence: 82%

Quantitative DY‐maleimide‐based proteomic 2‐DE‐labeling strategies using human skin proteins

et al. 2009

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Sensitive differential proteomic analysis is challenging and often limited by distinct labeling or tagging strategies. In this study, we have examined the sensitivity, linearity, and photophysical properties of novel protein labeling DY-maleimide dyes (DY-505-MAL, DY-555-MAL and DY-635-MAL). All MS compatible DY-maleimide dyes exhibited excellent emission spectra, high sensitivity, and high linearity, when applied to standard 1-DE protein analysis. Correspondingly, 2-DE analysis of DY-635-MAL or DY-505-MAL maximal-labeled human keratinocyte proteins displayed remarkably high sensitivity. Compared with a standard fluorescent protein stain, DY-635-MAL or DY-505-MAL 2-DE analysis demonstrated equally high spot quality with an overall increase in the number of spots detectable (up to threefold higher;41000 spots/gel). However, as determined with a FLA-5100 imaging system, comparative MultiGauge, and Delta2D analysis, not all DY-maleimide dyes possessed DIGE compatible fluorescent emission properties. However, DY-505-MAL and DY-635-MAL were found to be suitable for more complex, time and gel intensive, focused multiplexing analyses. Notably -as demonstrated with allergen-stimulated human skin proteins -defined, singular DY-maleimide dye protein labeling (SDPL) allows high quality, time saving, simple, and reliable differential proteomic examination.

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“…Each sample is initially alkylated with either unlabeled NEM or IAC, reduced, and fluorescently labeled with either green Cy3-or red Cy5-maleimide to code each sample (57). Infrared maleimide-based probes DY-680 and DY-780 have also been used to improve the sensitivity of detection (58). After labeling, the samples are combined and separated by 2D-gel electrophoresis.…”

Section: Redox Proteomicsmentioning

confidence: 99%

Regulatory Control or Oxidative Damage? Proteomic Approaches to Interrogate the Role of Cysteine Oxidation Status in Biological Processes

Held

Gibson

2012

Molecular & Cellular Proteomics

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Oxidation is a double-edged sword for cellular processes and its role in normal physiology, cancer and aging remains only partially understood. Although oxidative stress may disrupt biological function, oxidation-reduction (redox) reactions in a cell are often tightly regulated and play essential physiological roles. Cysteines lie at the interface between these extremes since the chemical properties that make specific thiols exquisitely redox-sensitive also predispose them to oxidative damage by reactive oxygen or nitrogen species during stress. Thus, these modifications can be either under reversible redox regulatory control or, alternatively, a result of reversible or irreversible oxidative damage. In either case, it has become increasingly important to assess the redox status of protein thiols since these modifications often impact such processes as catalytic activity, conformational alterations, or metal binding. To better understand the redox changes that accompany protein cysteine residues in complex biological systems, new experimental approaches have been developed to identify and characterize specific thiol modifications and/or changes in their overall redox status. In this review, we describe the recent technologies in redox proteomics that have pushed the boundaries for detecting and quantifying redox cysteine modifications in a cellular context. While there is no one-size-fits-all analytical solution, we highlight the rationale, strengths, and limitations of each technology in order to effectively apply them to specific biological questions. Several technological limitations still remain unsolved, however these approaches and future developments play an important role toward understanding the interplay between oxidative stress and redox signaling in health and disease. Molecular & Cellular Proteomics 11: 10.1074/mcp.R111.013037, 1-14, 2012. CYSTEINE: AN UNCOMMONLY REACTIVE AMINO ACIDThe nucleophilic sulfur atom allows cysteines to undergo a broad range of chemical modifications. These modifications include redox reactions, lipid acylation, and metal binding motifs that are important for protein structure, localization, regulation, and catalysis. Metal binding and oxidation play a role in protein structure through iron-sulfur (Fe-S) clusters, zinc fingers (ZF) 1 , and disulfide bonding, among others. Catalytic cysteines are essential to the function of numerous enzymes such as the E1 and E2 ligases of ubiquitin and ubiquitin-like proteins; the HECT domain of ubiquitin E3 ligases; SENP family sumo proteases; the tyrosine phosphatases protein phosphatase 1b (PTP1b) and PTEN; and many others including antioxidants in the thioredoxin, glutaredoxin, and peroxiredoxin families.Multiple thiol chemistries can converge to regulate the function of individual cysteines in a biological context. An example of the interconnection between the catalytic and redox properties of a cysteine is found in the cysteine-dependent aspartate-directed protease family of caspases. Essential to apoptosis, caspases are cysteine pr...

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Differential protein labeling with thiol‐reactive infrared DY‐680 and DY‐780 maleimides and analysis by two‐dimensional gel electrophoresis

Cited by 17 publications

References 8 publications

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Quantitative DY‐maleimide‐based proteomic 2‐DE‐labeling strategies using human skin proteins

Regulatory Control or Oxidative Damage? Proteomic Approaches to Interrogate the Role of Cysteine Oxidation Status in Biological Processes

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