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Kathryn S. Lilley - One of the best experts on this subject based on the ideXlab platform.
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Difference Gel Electrophoresis DIGE
Springer Protocols Handbooks, 2009Co-Authors: David B. Friedman, Kathryn S. LilleyAbstract:Proteomics offers powerful technologies to assist in the discovery of targets for novel therapeutic agents, by allowing the investigation of changes in protein state between control and diseased tissue and biofluids. Difference Gel Electrophoresis coupled with mass spectrometry (DIGE/MS) is a technology used within proteomics that has demonstrated technical robustness and associated statistical confidence to enable successful identification of therapeutic targets.
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Chapter 19 Measuring redox changes to mitochondrial protein thiols with redox Difference Gel Electrophoresis (redox-DIGE).
Methods in enzymology, 2009Co-Authors: Thomas R. Hurd, Kathryn S. Lilley, Andrew M. James, Michael P. MurphyAbstract:Low levels of reactive oxygen and nitrogen species (ROS and RNS) produced by the mitochondrial respiratory chain and ROS and RNS from other sources act as redox signals by oxidizing thiols on specific proteins. Because these thiol modifications occur on a relatively small number of proteins in the absence of bulk thiol changes, it is necessary to use sensitive methods to discover them. Recently, a number of methods have been developed to help facilitate the identification and characterization of redox‐sensitive thiol proteins. In this chapter we describe one such method, redox Difference Gel Electrophoresis (redox‐DIGE), in which oxidized thiol proteins in redox‐challenged samples are labeled with a thiol‐reactive fluorescent tag and compared with those in control samples labeled with a different tag on the same 2‐D Gel. This enables the sensitive detection of redox‐sensitive thiol proteins by measuring changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. With this method we have been able to identify several mitochondrial proteins whose thiol state and activity are altered by low levels of ROS from the respiratory chain, which may be an important and unexplored mode of mitochondrial redox signaling. Importantly, this method is not only applicable to studies in isolated mitochondria but can also be applied to more complicated systems such as intact cells and perhaps even whole organisms.
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Optimizing the Difference Gel Electrophoresis (DIGE) technology.
Methods in molecular biology (Clifton N.J.), 2008Co-Authors: David B. Friedman, Kathryn S. LilleyAbstract:Difference Gel Electrophoresis (DIGE) technology has been used to provide a powerful quantitative component to proteomics experiments involving 2D Gel Electrophoresis. DIGE combines spectrally resolvable fluorescent dyes (Cy2, Cy3, and Cy5) with sample multiplexing for low technical variation, and uses an internal standard methodology to analyze replicate samples from multiple experimental conditions with unsurpassed statistical confidence for 2D Gel-based differential display proteomics. DIGE experiments can facilely accommodate sufficient independent (biological) replicate samples to control for the large interpersonal variation expected from clinical samples. The use of multivariate statistical analyses can then be used to assess the global variation in a complex set of independent samples, filtering out the noise from technical variation and normal biological variation thereby focusing on the underlying variation that can describe different disease states. This chapter focuses on the design and implementation of the DIGE methodology employing the use of a pooled-sample internal standard in conjunction with the minimal CyDye chemistry. Notes are also provided for the use of the alternative saturation labeling chemistry.
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Detection of Reactive Oxygen Species-sensitive Thiol Proteins by Redox Difference Gel Electrophoresis IMPLICATIONS FOR MITOCHONDRIAL REDOX SIGNALING
The Journal of biological chemistry, 2007Co-Authors: Thomas R. Hurd, Kathryn S. Lilley, Tracy A. Prime, Michael E. Harbour, Michael P. MurphyAbstract:Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox Difference Gel Electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional Gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.
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Identification of clock genes using Difference Gel Electrophoresis.
Methods in molecular biology (Clifton N.J.), 2007Co-Authors: Natasha A. Karp, Kathryn S. LilleyAbstract:Proteomics is the study of the complete set of proteins encoded by the genome. The study of the proteome involves the investigation of changes in protein abundance, localization, involvement in multiprotein complexes, and detection of different protein isoforms and posttranslational modifications under defined conditions, such as the circadian cycle. This type of approach complements comparative gene expression studies providing additional information with respect to posttranscriptional processing. One of the key techniques used to study the proteome is two-dimensional Gel Electrophoresis. This technique has the ability to separate complex protein mixtures with high resolution. A significant improvement in this technology has been development of Difference Gel Electrophoresis. Here, proteins are first labeled with one of three spectrally resolvable fluorescent cyanine dyes before being separated in two dimensions according to their charge and size, respectively. Multiplexing can accurately and reproducibly quantify protein expression across multiple Gels. A multiple-Gel approach allows the detection of differentially expressed protein spots using statistical methods to compare expression across different experimental groups. The proteins can be subsequently identified by mass spectrometric methods. This approach now allows more complex experimental designs, such as the time course experiments essential to the study of circadian rhythms.
Tadashi Kondo - One of the best experts on this subject based on the ideXlab platform.
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Calreticulin as A Novel Potential Metastasis-Associated Protein in Myxoid Liposarcoma, as Revealed by Two-Dimensional Difference Gel Electrophoresis
MDPI AG, 2019Co-Authors: Takashi Tajima, Fusako Kito, Akihiko Yoshida, Akira Kawai, Tadashi KondoAbstract:Myxoid liposarcoma (MLS) is a mesenchymal malignancy. To identify innovate seeds for clinical applications, we examined the proteomes of primary tumor tissues from 10 patients with MLS with different statuses of postoperative metastasis. The protein expression profiles of tumor tissues were created, and proteins with differential expression associated with postoperative metastasis were identified by two-dimensional Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry. The validation was performed using specific antibodies and in vitro analyses. Using 2D-DIGE, we observed 1726 protein species and identified proteins with unique expression levels in metastatic MLS. We focused on the overexpression of calreticulin in metastatic MLS. The higher expression of calreticulin was confirmed by Western blotting, and gene silencing assays demonstrated that reduced expression of calreticulin inhibited cell growth and invasion. Our findings suggested the important roles of calreticulin in MLS metastasis and supported its potential utility as a prognostic biomarker in MLS. Further investigations of the functional properties of calreticulin and other proteins identified in this study will improve our understanding of the biology of MLS and facilitate novel clinical applications
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Cancer biomarker development and two-dimensional Difference Gel Electrophoresis (2D-DIGE).
Biochimica et biophysica acta. Proteins and proteomics, 2018Co-Authors: Tadashi KondoAbstract:Cancer results from the accumulation of genomic alterations. As the genome is functionally translated to the proteome and regulates tumor cell behavior, proteomics studies are expected to further the current understanding of the molecular mechanisms underlying carcinogenesis and cancer progression. Biomarkers are potential tools to classify cancers for therapy, predict responses to treatments, and support treatment-related decision-making. Biomarker development has been actively pursued in oncology by proteomic approaches. Two-dimensional Difference Gel Electrophoresis (2D-DIGE) is a proteomics technique based on two-dimensional polyacrylamide Gel Electrophoresis (2D-PAGE). In 2D-DIGE, protein samples are labeled with distinct fluorescent dyes before fractionation via 2D-PAGE. 2D-DIGE offers advantages to identify biomarker candidates, including reproducibility, high sensitivity, comprehensiveness, and high throughput. 2D-DIGE has contributed to the establishment of tissue biomarkers, which potentially facilitate precision medicine. 2D-DIGE is thus expected to yield major advancements in cancer biomarker identification and development.
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Laser microdissection and two-dimensional Difference Gel Electrophoresis reveal the role of a novel macrophage-capping protein in lymph node metastasis in gastric cancer.
Journal of proteome research, 2013Co-Authors: Hiroshi Ichikawa, Tatsuo Kanda, Shin-ichi Kosugi, Yasuyuki Kawachi, Hiroki Sasaki, Toshifumi Wakai, Tadashi KondoAbstract:To reveal the proteomic background of lymph node metastasis (LNM) in gastric cancer, we performed a proteomic study of tumor and matched nontumor tissues obtained from surgically resected specimens of 22 patients with or without LNM. Using laser microdissection, we recovered specific populations of tumor and nontumor cells. We used two-dimensional Difference Gel Electrophoresis with a large format Electrophoresis apparatus to obtain protein expression profiles consisting of 3228 protein spots, and we classified them according to their expression pattern. We found that macrophage-capping protein (CapG) was up-regulated in the tumor tissues of patients with LNM, whereas it showed an equivalent expression level between nontumor and tumor tissues of patients without LNM. It was reported that CapG associated with invasion and metastasis in various malignancies. However, CapG was not investigated in gastric cancer until our study. Western blotting of the laser microdissected tissue samples confirmed up-regulati...
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Abstract 2490: Novel application for pseudopodial proteomics using excimer laserablation and two-dimensional Difference Gel Electrophoresis.
Cancer Chemistry, 2013Co-Authors: Takahiro Mimae, Tadashi Kondo, Akihiko Ito, Man Hagiyama, Morihito OkadaAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial microprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional Difference Gel Electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than 4-fold Differences and p value less than 0.05). The protein enrichment depended on the pore size. Mass spectrometric protein identification revealed 46 pseudopodia-localizing proteins. The localization of novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin was confirmed using immunohistochemical examinations. The previous studies demonstrated that these four proteins may function in the cell migration process. In addition, RAB1A was indicated to play an important role in forming and extending pseudopodial microprocesses in human breast cancer cells by overexpression and knockdown experiments. This method will provide insights into the molecular details of pseudopodia and further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. Citation Format: Takahiro Mimae, Akihiko Ito, Man Hagiyama, Tadashi Kondo, Morihito Okada. Novel application for pseudopodial proteomics using excimer laserablation and two-dimensional Difference Gel Electrophoresis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2013-2490
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Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional Difference Gel Electrophoresis
Laboratory investigation; a journal of technical methods and pathology, 2012Co-Authors: Akihiko Ito, Takahiro Mimae, Man Hagiyama, Morihito Okada, Ying-shan-zhu Yamamoto, Jun Nakanishi, Masaoki Ito, Yoichiroh Hosokawa, Yoshinori Murakami, Tadashi KondoAbstract:We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads to a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial microprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional Difference Gel Electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than fourfold Differences and P-value of
Sandrine Guillou - One of the best experts on this subject based on the ideXlab platform.
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Two-dimensional fluorescence Difference Gel Electrophoresis analysis of Listeria monocytogenes submitted to a redox shock.
Journal of Proteomics, 2013Co-Authors: Maria Ignatova, Blandine Guével, Emmanuelle Com, Nabila Haddad, Albert Rossero, Philippe Bogard, Hervé Prévost, Sandrine GuillouAbstract:The influence of redox alteration on the growth and proteomic pattern of Listeria monocytogenes was investigated. A redox shock was induced in cultures by addition of 3mM ferricyanide (FeCN) and 6mM dithiothreitol (DTT) to increase or to decrease respectively the redox potential naturally occurring at the beginning of growth. In both conditions, the reducing and oxidizing redox shock had a strong influence, decreasing the maximum growth rate by half compared to a control culture. The proteomic analysis of L. monocytogenes performed by two-dimensional Difference Gel Electrophoresis (2D-DIGE) exhibited twenty-three proteins differentially expressed (P
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two dimensional fluorescence Difference Gel Electrophoresis analysis of listeria monocytogenes submitted to a redox shock
Journal of Proteomics, 2013Co-Authors: Maria Ignatova, Blandine Guével, Emmanuelle Com, Nabila Haddad, Albert Rossero, Philippe Bogard, Hervé Prévost, Sandrine GuillouAbstract:The influence of redox alteration on the growth and proteomic pattern of Listeria monocytogenes was investigated. A redox shock was induced in cultures by addition of 3mM ferricyanide (FeCN) and 6mM dithiothreitol (DTT) to increase or to decrease respectively the redox potential naturally occurring at the beginning of growth. In both conditions, the reducing and oxidizing redox shock had a strong influence, decreasing the maximum growth rate by half compared to a control culture. The proteomic analysis of L. monocytogenes performed by two-dimensional Difference Gel Electrophoresis (2D-DIGE) exhibited twenty-three proteins differentially expressed (P<0.05), among these, many were oxidoreductases, and proteins involved in cellular metabolism (glycolysis, protein synthesis), detoxification (kat) or adhesion (Lmo1634).
Jonathan S. Minden - One of the best experts on this subject based on the ideXlab platform.
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Two-dimensional Difference Gel Electrophoresis.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Jonathan S. MindenAbstract:Two-dimensional Difference Gel Electrophoresis (2D DIGE) is a modified form of 2D Electrophoresis (2D E) that allows one to compare two or three protein samples simultaneously on the same Gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during Electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With conventional imaging systems, DIGE is capable of reliably detecting as little as 0.2 fmol of protein, and protein Differences down to ± 15%, over a ~10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 days to complete. We have further improved upon 2D DIGE by introducing in-Gel equilibration to improve protein retention during transfer between the first and second dimensions of Electrophoresis and by developing a fluorescent Gel imaging system with a millionfold dynamic range.
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Two-Dimensional Difference Gel Electrophoresis (2D DIGE)
Methods in Cell Biology, 2012Co-Authors: Jonathan S. MindenAbstract:Abstract Two-dimensional Difference Gel Electrophoresis (2D DIGE) is a modified form of 2D Electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same Gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during Electrophoresis. Proteins that are common to the samples appear as “spots” with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, DIGE is capable of reliably detecting as little as 0.2 fmol of protein, and protein Differences down to ±15%, over an approximately 10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2–3 days to complete.
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Difference Gel Electrophoresis.
Electrophoresis, 2009Co-Authors: Jonathan S. Minden, Susan R Dowd, Helmut E Meyer, Kai StühlerAbstract:Difference Gel Electrophoresis (DIGE) was invented to circumvent the inherent variability of 2-DE. This variability is a natural consequence of separating thousands of proteins over a large space, such as a 15 x 20 cm slab of polyacrylamide Gel. The originators of 2-DE envisioned being able to compare cancerous cells and normal cells to understand what makes these cells different. Gel-to-Gel variability made this an extremely difficult task. We reasoned that if both samples could be run on the same Gel, then the inherent variability would be obviated. Thus, we created matched sets of fluorescent dyes that allows one to compare two or three protein samples on a single Gel. In the 12 years since the description of DIGE first appeared in Electrophoresis, this founding paper has been cited over 660 times. This review highlights some of the improvements and applications of DIGE. We hope these examples are illustrative of what has been done and where the field is headed.
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Comparative proteomics and Difference Gel Electrophoresis.
BioTechniques, 2007Co-Authors: Jonathan S. MindenAbstract:The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional Electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference Gel Electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE Gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE.
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Lights, Camera, Action! systematic variation in 2-D Difference Gel Electrophoresis images.
Electrophoresis, 2007Co-Authors: Kimberly F. Sellers, Jonathan S. Minden, Surya Viswanathan, Jeffrey C. Miecznikowski, William F. EddyAbstract:2-D Difference Gel Electrophoresis (DIGE) circumvents many of the problems associated with Gel comparison via the traditional 2-DE approach. DIGE's accuracy and precision, however, is compromised by the existence of other significant sources of systematic variation, including that caused by the apparatus used for imaging proteins (location of the camera and lighting units, background material, imperfections within that material, etc.). Through a series of experiments, we estimate some of these factors, and account for their effect on the DIGE experimental data, thus providing improved estimates of the true relative protein intensities. The model presented here includes 2-DE images as a special case.
David B. Friedman - One of the best experts on this subject based on the ideXlab platform.
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Difference Gel Electrophoresis DIGE
Springer Protocols Handbooks, 2009Co-Authors: David B. Friedman, Kathryn S. LilleyAbstract:Proteomics offers powerful technologies to assist in the discovery of targets for novel therapeutic agents, by allowing the investigation of changes in protein state between control and diseased tissue and biofluids. Difference Gel Electrophoresis coupled with mass spectrometry (DIGE/MS) is a technology used within proteomics that has demonstrated technical robustness and associated statistical confidence to enable successful identification of therapeutic targets.
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Optimizing the Difference Gel Electrophoresis (DIGE) technology.
Methods in molecular biology (Clifton N.J.), 2008Co-Authors: David B. Friedman, Kathryn S. LilleyAbstract:Difference Gel Electrophoresis (DIGE) technology has been used to provide a powerful quantitative component to proteomics experiments involving 2D Gel Electrophoresis. DIGE combines spectrally resolvable fluorescent dyes (Cy2, Cy3, and Cy5) with sample multiplexing for low technical variation, and uses an internal standard methodology to analyze replicate samples from multiple experimental conditions with unsurpassed statistical confidence for 2D Gel-based differential display proteomics. DIGE experiments can facilely accommodate sufficient independent (biological) replicate samples to control for the large interpersonal variation expected from clinical samples. The use of multivariate statistical analyses can then be used to assess the global variation in a complex set of independent samples, filtering out the noise from technical variation and normal biological variation thereby focusing on the underlying variation that can describe different disease states. This chapter focuses on the design and implementation of the DIGE methodology employing the use of a pooled-sample internal standard in conjunction with the minimal CyDye chemistry. Notes are also provided for the use of the alternative saturation labeling chemistry.
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Proteomic investigation of human burn wounds by 2D-Difference Gel Electrophoresis and mass spectrometry
The Journal of surgical research, 2007Co-Authors: Alonda C. Pollins, David B. Friedman, Lillian B. NanneyAbstract:Background In humans, thermal cutaneous injury represents a serious traumatic event that induces a host of dynamic alterations. Unfortunately the molecular mechanisms that underlie these serious perturbations remain poorly understood. We applied a global analysis method to identify dynamically changing proteins within the burn environment, which could eventually become biomarkers or targets for treatment. Materials and methods Protein extracts of normal/unwounded skin and burn wounds were assayed by 2D-Difference Gel Electrophoresis (DIGE), a proteomic technology by which abundance levels of intact proteins (including isoforms) were simultaneously quantified from multiple samples with statistical confidence. Through unsupervised multivariate principal component analysis, protein expression patterns from individual samples were appropriately clustered into their correct temporal healing periods grouped into postburn periods of 1–3 days, 4–6 days, or 7–10 days after injury. Forty-six proteins were subsequently selected for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Results Proteins identified with differential temporal patterns of expression included predictable cytoskeletal proteins such as vimentin, and keratins 1, 5, 6, 16, and 17. Other candidate proteins with potential involvement in healing included heat shock protein 90, members of the serpin family (Serpin B1, SCCA1 and -2), haptoglobin, Gelsolin, eIF4A1, IQGAP1, and translationally controlled tumor protein. Conclusions We have used the combined technique, DIGE/mass spectrometry, to capture new insights into cutaneous responses to burn trauma and subsequent processes of early wound healing in humans. This pilot study provides a proteomic snapshot of temporal events that can be used to weave together the interconnected processes that define the response to serious cutaneous injury.
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Quantitative Proteomics for Two-Dimensional Gels Using Difference Gel Electrophoresis
Methods in molecular biology (Clifton N.J.), 2007Co-Authors: David B. FriedmanAbstract:Difference Gel Electrophoresis (DIGE) technology provides a powerful quantitative component to proteomics experiments involving two-dimensional (2D) Gel Electrophoresis. DIGE allows for the detection of subtle changes in protein abundance with statistical confidence while controlling for Gel-to-Gel variation, as well as additional variation that is non-biological in origin (e.g., sample preparation error, normal variation in a system). Samples are differentially labeled with spectrally resolvable fluorescent dyes (Cy2, Cy3, and Cy5) and co-resolved for direct quantification within the same 2D Gel. Increased statistical confidence is obtained when combining experimental repetition with internal standards such that independent replicate measurements from single- and multivariable analyses can be intercompared with a relatively small number of coordinated DIGE Gels.
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proteome analysis of human colon cancer by two dimensional Difference Gel Electrophoresis and mass spectrometry
Proteomics, 2004Co-Authors: David B. Friedman, Salisha Hill, Jeffrey W Keller, Nipun B Merchant, Shawn Levy, Robert J Coffey, Richard M CaprioliAbstract:Two-dimensional Difference Gel Electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D Gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to Gel-to-Gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.