Fluorescence 2-D difference gel electrophoresis (DIGE) uses spectrally resolvable dyes to label protein samples prior to 2-D electrophoresis. By using different fluorescent dyes to separately label protein samples multiple samples can be co-separated and visualized on a single 2-D gel. Differences between samples are resolved using image analysis software such as DeCyder 2D. This fluorescent multiplexing approach is compatible with mass spectrometry and overcomes many of the disadvantages of traditional 2-D analyses. A broad dynamic range provides more accurate quantitative data than traditional 2-D silver staining techniques while rapid image overlay simplifies image analysis and improves comparative accuracy.

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Proteomic study is an effective approach in the disease research, because proteomic aberrations should exist behind any type of diseases and it is proteome that directly regulate the disease phenotypes. Two-dimensional difference gel electrophoresis (2D-DIGE) is a high performance proteomic technology. Although it is based on two-dimensional gel electrophoresis (2D-PAGE), owing to its multiplex detection system, it solves many drawbacks of classical 2D-PAGE. 2D-DIGE allows obtain the quantitative protein expression profiles across many clinical specimens in a reproducible and high-throughout manner. 2D-DIGE with high sensitive fluorescent dyes enables the proteomic study on the laser microdissected tissues, and thus accurate proteomics can be achieved using 2D-DIGE. Mass spectrometry can identify the proteins corresponding to any spots observed by 2D-DIGE and we can utilize the gene and literature database to interpret the proteomic data. Bioinformatic approach can determine the proteomic signatures responsible for the important clinico-pathological features and identify a small number of key proteins, which will be candidates for disease markers and therapeutic targets. Combination between 2D-DIGE, mass spectrometry and bioinformatics approach will be a powerful tool for disease proteomics. The efforts to understand the overall feature of proteome by bioinformatics approach to 2D-DIGE data, together with the integrated information of the individual proteins identified by 2D-DIGE, will give us novel molecular backgrounds of the diseases. The proteome database should facilitate the use of the wealth of abundant information. The data integration between genome, transcriptome and proteome is also important approach to understand the background of proteomic aberrations in diseases.

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We have attempted to separate a minimal amount of proteins of glomeluli isolated from biopsy tissues of human kidney on large format 2-DE gels by exploiting extremely sensitive fluorescent dyes (Cy3- and Cy-5 saturation dye). Only 2.5μg glomerular proteins corresponding to 2-3 glomeruli, gave highly resolved 2-DE profiles, suggesting the applicability of 2-DE to analyze glomerulus proteome in biopsy specimens. We propose a platform for highly quantitative analysis of differential protein expression in the glomerulus in health and disease with the saturation dye as a detection tool.

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A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) offers advantages over the more widely used immobilized pH gradient 2-DE for separating high-molecular-mass proteins (100-500kDa) and for having a higher loading capacity (1.5mg in total). We have applied the Fluorescent 2-D differential gel electrophoresis (2-D DIGE) to our agarose 2-DE system. This allowed us to see clear differences in the 2-DE patterns from liver extracts of a diabetic model Otsuka Long-Evans Tokushima Fatty (OLETF) and its control Long-Evans Tokushima Otsuka (LETO) rats including changes in the amounts of several proteins larger than 100kDa. The combined method would increase its power to detect changes in disease proteomics.

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Two-dimensional differential in-gel electrophoresis technology (2D DIGE) technique is highly useful for differential analysis of protein spots in two-dimensional differential gels. Utilizing this technique, we attempted to search for diabetes-related drug targets and biomarkers. In human hepatoma cell line, HepG2, we analyzed secretome in the presence or absence of a Liver X receptor agonist, TO-901317, and identified one of the up-regulated proteins in response to LXR activation as apolipoprotein E. We also evaluated nuclear proteome of cultured cells overexpressing insulin receptor substrate proteins, in which insulin-stimulated cell cycle progression is differentially regulated, and the gel pattern indicated that insulin-induced phosphorylation of a nuclear protein may be impaired in cells overexpressing cell cycle-suppressive insulin receptor substrate-3. In addition, to search for urinary markers of diabetic nephropathy using 2D DIGE, we analyzed urine samples in which most abundant proteins were removed by immunoaffinity depletion. These findings indicate that the 2D DIGE-based approach is useful for the discovery of disease-specific drug targets and diagnostic biomarkers.

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We introduce a novel fluorescence resonance energy transfer (FRET) system for the detection of a phosphorylated molecule such as a phosphopeptide using a phosphate-binding tag molecule, Zn(II)-Phos-tag (1,3-bis[bis (pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) attached with a 7-amino-4-methylcoumarin-3-acetic acid (AMCA). 5-Carboxyfluorescein (FAM)-labeled phosphopeptides and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, emission at 520 nm) and AMCA (a fluorescent donor, excitation at 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn(II)-Phos-tag captured specifically the FAM-labeled phosphopeptide to form a stable 1:1 complex, resulting in efficient FRET. After the FAM-labeled phosphopeptide was dephosphorylated with alkaline phosphatase, the FRET disappeared. Using this FRET system, we demonstrated the detection of the time-dependent reversible phosphorylation of the FAM-labeled substrate peptide. The Phos-tag-based FRET system has the following major advantages: i) The real-time analysis of the reversible phosphorylation reaction is possible without multiple samplings, ii) the analysis requires a simple procedure just using two solutions of AMCA-labeled Phos-tag and a FAM-labeled compound, and iii) the system would be useful for the reliable and comprehensive phosphorylation assays for various phosphopeptides containing phosphoserine, phosphothreonine, or phosphotyrosine, in vitro. Thus, the principle of this system would be applied to high-throughput kinase/phosphatase profiling, measurement of enzyme activity, and determination of an activator or an inhibitor.

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Recent advances on analytical technology of proteomics offer exciting opportunities to find novel and multiple biomarkers. Among proteomic procedures for differential analysis developed until today, two-dimensional differential gel electrophoresis (2D-DIGE) is one of the most useful techniques on analyzing the proteins containing their modified forms. Using this 2D-DIGE, we carried out a search for the disease-associated proteins linking to the potential diagnostic biomarkers for a highly malignant type in human ovarian cancer, clear cell adenocarcinoma (CCA).
In this study, we first performed a differential analysis using human ovarian cultured cell lines OVISE and OVTOKO as CCA cell lines in comparison with MCAS as a control cell line. Then the proteins characteristically expressed in CCA cell lines were identified by mass spectrometry. As a result of this analysis, some of the identified proteins were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that some of the identified proteins were expressed differentially in actual tissues as well as in the CCA culture cells. The results from this investigation show the effectiveness of a proteomic approach to identify expressed proteins that are characteristic for particular cancers, which may eventually serve as diagnostic markers or therapeutic targets in CCA.

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The pyridylamination method was originally described in 1978 as a means of analyzing glycan structures with high-sensitivity. Subsequently, the method has been applied to structure analyses of glycans including glycosidase digestion, 2D-mapping by various kinds of HPLC's, partial acetolysis, Smith degradation, methylation analysis, nuclear magnetic resonance, mass spectrometry, and affinity assay for lectins. Glycans on glycoconjugates are liberated by hydrazinolysis followed by N-acetylation. Reducing ends of the released glycans are tagged with 2-aminopyridine by reductive amination. Pyridylamino (PA-) derivatives of glycans with fluorescence and a positive charge have the following advantages: 1. high sensitivity in detection; 2. excellent separation in reversed phase HPLC; 3. high chemical stability under the conditions for structure elucidation; 4. applicable to many authentic methods for glycan analysis.

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We used fluorescein isothiocyanate (FITC) as a stain for the detection of two-dimensionally separated proteins. Human serum proteins were pre-labeled with FITC by mixing serum and a FITC solution at 0°C and pH 9.2 for 24 hours and subjected to two-dimensional electrophoresis in the absence of denaturing agents. The distribution of labeled proteins on the slab gel could be observed during and just after the electrophoretic run by irradiation with UV light.
When the FITC concentration was below 3.5mM in serum-FITC mixture, the mobilities of serum proteins did not differ from those of the native proteins. By controlling the FITC concentration, selective labeling of specific serum proteins was possible

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Affinophoresis is a type of affinity electrophoresis in which an affinophore, a conjugate of an affinity ligand and a multiply charged soluble matrix, causes a mobility change in proteins that have specific affinity for the ligand. This technique enables, not only the separation of a specific molecule, but also the analysis of specific interaction between biomolecules. The affinity constant between fluorescence-labeled pea lectin and affinophores bearing mannoside as an affinity ligand, was determined by capillary electrophoresis using laser-induced-fluorescence detection. Although the labeling of the protein caused microheterogeneity as evidenced by electrophoretic mobility, the affinity constant determined for the main peak in the electropherogram of the labeled lectin agreed with those obtained for unlabeled lectin by the same type of analysis using ultraviolet-absorption detection. As a result of the use of the fluorescence detection, the consumption of the protein sample is greatly reduced to several pg per run.

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Human granulocytic anaplasomsis and human monocytic ehrlichiosis are tick-borne emerging infectious diseases which are caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis. These bacteria are obligatory intracellular parasites with tropism for hematopoietic cells. Here, we introduced analysis of protein expression profiles of HL-60 host cells in the response to infection with those bacteria by proteomic approach using fluorescence-prelabeled two dimensional difference gel electrophoresis to clarify the molecular mechanisms of the intracellular parasitism. Three experiments were designed for this purpose: differential analysis of infection at late (i) or early (iii) stages, and time-course analysis postinfection (ii). The detailed technical approaches, advantage of this procedure, and technical improvements desired were described and discussed.

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骨軟部肉腫は，骨や筋肉などの間質より発生する悪性腫瘍である．小児や若年成人に発生し予後不良な転機をたどることが多く，予後改善のための研究は臨床的に重要である．また骨軟部肉腫は病理学的に50種類以上の組織型に複雑に分類されている．組織型によって予後や治療感受性が異なるため，骨軟部肉腫の治療成績改善のためには鑑別診断や予後予測，治療奏効性予測などのバイオマーカーの開発が必要である．我々は，主に蛍光二次元電気泳動法を用いて，骨軟部肉腫のタンパク質発現を網羅的に解析し，臨床応用を目指したバイオマーカーの開発を行っている．本稿では，消化管間質腫瘍の予後予測マーカーの開発とその発現検証試験の成果，および骨肉腫の化学療法奏効性予測マーカーの開発について紹介する．これらのバイオマーカーにより新たな治療戦略の策定および個別化医療の推進が可能となり，患者のQOL・治療成績改善に大きく寄与する事が期待される．

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In many applications, an understanding of differentially expressed genes in different tissues or owing to an applied stimulus is important. However, the wide use of polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR) and differential display (DD) has shown that using radioisotopes is still a problem. Although a technique has been developed that avoids some of the disadvantages, the use of radioisotopes for band detection still limits various applications. It has been improved this technique for analyzing differentially expressed mRNA by resolving the amplified products using fluorescent-labeling as a non-radioisotope. Our modified method allows the improvement of identification on differentially expressed bands with a high accuracy. This technique with a non-radioisotope representation may be possible to perform PCR-DD analysis with many applications.

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A sensitive and separative fluorometric analysis of alkaline phosphatase isoenzyme was described. Electrophoretical separation of alkaline phosphatase isoenzyme was based on acetate gel (Cellogel) electrophoresis with the discontinuous buffer system, and the locarization of alkaline phosphatase isoenzyme was fluorometrically visualized with naphthol ASMX-phosphate as substrate. The liberated naphthol ASMX which has a strong fluorescence at 505nm on the acetate gel were scanned by scanning fluorophotometer, and the peak areas obtained from scannings were proportional to the activities of each separated isoenzyme. The standard curves were linear in the rang of 5 to 640 U/1. The coefficient of variation of this method as a quantitative analysis of enzyme separated was 3 to 5 per cent in the same membrane and 8 to 10 per cent in the different membranes.
Using this method, the kinetical inhibition studies of alkaline phosphatase isoenzymes were easilly carried out by the substrate solution containing different concentration of inhibitors, and the kinetical values of the enzymes in the solution of enzyme mixture were clearly coincided with the kinetical values obtained from the purified enzymes. The usefullness and application of this sensitive microtechniques in clinical examination was studied and discussed.

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DNA profiling has become a standard technique in criminal investigations due to its ability to obtain results from any biological material. PCR-based DNA typing systems have also enabled analyses of DNA obtained from highly decomposed human remains. Based on the length and genomic distribution of the core repeat sequences, two systems are classified as minisatellites and microsatellites (or short tandem repeats). In particular, short tandem repeats (STRs) are abundant classes of polymorphic loci dispersed throughout the entire genome. Each STR locus usually consists of a limited number of the core repeats of 2-5 basepairs, which can easily be resolved by analytical gel electrophoresis. In combination with the use of fluorescence-labelled primers employing different fluorescent dyes, STR loci with overlapping or identical allele size ranges can be analyzed together in the same lane of polyacrylamide gel, thus providing a rapid and accurate method for human identification. Automated DNA profiling by fluorescence-based technology, as well as its application, have been studied and validated for use in forensic laboratory routine.

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FACE (fluorophore-assisted carbohydrate electrophoresis) is a method for the separation of fluorophore-labeled saccharides by the use of polyacrylamide gel electrophoresis. The method is characterized by high resolution, high sensitivity, and ease of use without expensive giant-sized machine such as mass spectrometry. It has been found to be useful for the analysis of oligosaccharides profiling of N-linked or O-linked glycans after releasing saccharides from glycoproteins and for sequencing oligosaccharides component. Judging from the importance of oligosaccharides in glycoproteins and the changes in oligosaccharides of cancer cell surface, the method described here is hopeful as a unique analysis tool in the field of basic medical science.

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