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Volume 51 (2003)
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- Issue 5 Pages 492-
- Issue 4 Pages 439-
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Volume 51, Issue 5
Displaying 1-11 of 11 articles from this issue
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REVIEW
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Toshitaka SATO, Yoshiya ODA, Takashi OWA, Takeshi NAGASU2003 Volume 51 Issue 5 Pages 492-498
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSThe completion of the human genome project brings a paradigm shift for drug discovery. It has been said that use of genomic information could accelerate the identification of new drug targets and the development of tailor-made drugs. Rising new technologies, such as transcriptomics (DNA microarray analysis), proteomics and bioinformatics, have made it possible to analyze a global expression of genes and proteins and compare their difference between normal and disease conditions. But it is also become evident that such flood of enormous data is not necessarily prerequisite for new drug discovery, especially for drug target identification. Chemical compounds with pharmacological activities appear to be one of the key tools to narrow down druggable targets on the basis of their interaction with cellular proteins and their effects on gene and protein expression. Here we show recent technologies that exploit chemical probes for identifying promising drug target proteins.View full abstractDownload PDF (238K)
REGULAR PAPERS
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Takuji NABETANI, Yo TABUSE, Akira TSUGITA2003 Volume 51 Issue 5 Pages 499-503
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSAnimal development proceeds through harmonized expression of many genes. A small free-living soil nematode, Caenorhabditis elegans, has many advantages as a model organism and has been used to study the genetic program, operating in animal development. In recent years, exhaustive measurement of mRNA level using DNA microarray have been carried out in various biological systems, including C. elegans and have provided valuable information about gene expression during development. On the other hand, it is often pointed out that analyses of protein level might be rather critical, since the function of a gene is accomplished by the expressed protein. To identify proteins which are expressed differentially during development and to delineate the correlation of mRNA and protein expression in C. elegans, we have conducted differential two-dimensional electrophoresis of protein samples from the embryo to adult animal. Differential image analysis of gel patterns revealed that many proteins were expressed stage-specifically and they were analyzed with MALDI-TOF mass-spectrometry. In this report, we show the result of ten proteins which expressed mainly either in the embryo or in other developmental stages besides the embryo. Although expression patterns of most of them were found to be correlated with those of mRNA level, the expression pattern of DIM-1 protein is inconsistent with that of mRNA. The discrepancy between protein and mRNA levels found in our limited scale of experiment further strengthen the notion that gene expression does not always parallel with the expression of the corresponding protein.View full abstractDownload PDF (243K) -
Posttranslational Modification of Liver Glutamine Synthetase in HepatomaYasuhiro KURAMITSU, Toshifusa TODA, Masaaki OKA, Kiwamu OKITA, Kazuyuk ...2003 Volume 51 Issue 5 Pages 504-508
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSMass spectrometric analysis of molecular diversity of human liver glutamine synthetase (GS) is described. An acidic isoform of GS, which is highly expressed in human well-differentiated hepatocellular carcinoma, and that caused by posttranslational modification of protein phosphorylation which was determined by post source decay (PSD) analysis using matrix assisted laser deionization/ionization-time of flight (MALDI-TOF) mass spectrometry. The peptide mass fingerprinting (PMF) of tryptic peptides of the GS isoform showed a loss of the signal of 899.5 Da corresponding a peptide of SASIRIPR and a gain of the signal of 1,059.5 Da which was submitted to PSD analysis. The PSD analysis showed the neutral loss by elimination of two phosphate groups which were supposed to be on serine residues of 899.5 Da peptide from serine 320 to arginine 327 in GS. The PMF followed by PSD analysis is useful for the determination of phosphorylation site(s) of proteins showing molecular heterogeneity.View full abstractDownload PDF (182K) -
Tadakazu MAEDA, Masamichi OH-ISHI, Takeshi UENO, Yoshio KODERA2003 Volume 51 Issue 5 Pages 509-515
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSOxidative stress is implicated in a broad variety of chronic and acute diseases, including such age-related diseases as diabetes. To understand at the protein level cellular damage caused by the stress, we developed a proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by two-dimensional gel electrophoresis with agarose gels in the first dimension. The method, being capable of analyzing high-molecular-mass proteins as large as myosin heavy chains (molecular mass: 200 kDa), was applied to detecting protein carbonyls in muscles of a diabetes model OLETF rat and a control LETO rat. A number of proteins, including mitochondrial ATP synthase β-chain, desmin, actin, and myosin, were found carbonylated. Our method would provide a means toward clarifying a comprehensive view of oxidative modifications of proteins during a long progression of age-related diseases and understanding the mechanism of the onset, progression, and complication of the diseases.View full abstractDownload PDF (644K) -
Masamichi OH-ISHI, Yoshio KODERA, Tadakazu MAEDA2003 Volume 51 Issue 5 Pages 516-523
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSWhile human plasma or human tissues would be major sample resources to be analyzed in this field, proteome analysis of human samples often made complicated results depending on patient's individuality and/or conditions of the patient. Experimental animal models with hereditary diseases would be substitutes of various human diseases. We would like to introduce our proteome analysis strategy with two examples listed below: (i) rdw rat with hereditary hypothyroidism and dwarfism, and (ii) OLETF rat with hereditary diabetes. With 2-DE with agarose gels in the first dimension (agarose 2-DE), being capable of analyzing high-molecular-mass proteins (>100 kDa), in combination with LC-MS, and/or detecting method of oxidatively-induced protein carbonyls, we will present a means toward understanding the mechanism of the onset, progression, and complication of these diseases.View full abstractDownload PDF (927K)
COMMENTARY
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Purification of Protein with Post-translational Modifications by Affinity ChromatographyMasaki MATSUMOTO, Keiichi NAKAYAMA2003 Volume 51 Issue 5 Pages 524-529
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSRecent advances in methods for protein identification by a combination of mass spectrometry and protein/DNA sequence database enabled to study of high throughput analysis of proteome. In addition to expression level, numerous characterics of the proteins such as cellular localization, complex formation, stability, and post-transcriptional modifications can be studied by proteomic approach. However, whole proteome analysis has limitation to identify proteins with low expression level. Here we present that our strategy for purification of proteins with post-translational modifications, such as ubiquitylation and phosphorylation by affinity chromatography. Using antibodies against these modification groups, modified proteins were effectively purified. After digestion by trypsin, resulting peptides were subjected to online LC-ESI-MS/MS analysis. In addition to known substrate proteins, novel substrate proteins were identified. Thus, affinity purification of proteins that have post. translational modifications is effective method for focused proteomics.View full abstractDownload PDF (283K)
TECHNICAL REPORTS
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Keigo BUNAI, Tadashi NEMOTO, Kunio YAMANE2003 Volume 51 Issue 5 Pages 530-537
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSTo improve sample preparation method for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) with the aim of proteomics, proteins separated by Tris/tricine SDS-PAGE were electroblotted onto PVDF membrane by a semi-dry discontinuous buffer system, visualized by staining with Coomassie Brilliant Blue, excised, digested with trypsin or lysC in the presence of 80% acetonitrile. Digested proteins were analyzed with MALDI-TOF MS. This method has several advantages over “in gel digestion” in terms of simple handling, sensitivity and time. We analyzed 105 fmol of Bacillus subtilis SecA protein and 300 to 500 fmol of standard proteins. We also analyzed three submembrane fractions of B. subtilis which had been solubilized stepwise using different mixtures of detergents from a washed cell membrane that was insoluble in 134 mM non-detergent sulfobetain solution. After two different 1D- and three different 2D-PAGEs followed by MALDI-TOF MS analysis, we identified 637 different proteins. Among them, 357 were predicted to be membrane proteins having certain or putative transmembrane segments (TMS) using the analysis by TopPred II algorithm, but 280 were soluble proteins having no TMS. On the basis of a bioinformatics analysis on B. subtilis genome data-base, 38 ABC trasnporter solute-binding proteins were predicted and 30 of the 38 were identified by this analysis. However, this improved method still have a limitation for the analysis of cell membrane proteins having more than 4 TMSs in their amino acid sequences.View full abstractDownload PDF (549K) -
Hiroya KUMAI, Naoe ISHII2003 Volume 51 Issue 5 Pages 538-541
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSProteome analysis is an effective means to identify molecules which have important roles in cells or organs. In this analysis, the electrophoresis is indispensable technology to separate proteins in details. However, the lateness of separation speed is a bottleneck for high throughput. We tried the electrophoresis with gels and buffers prepared by three type waters. As a comparison of the water, the purity shows possibility to contribute to acceleration of the electrophoresis speed. On the other hand, MALDI-TOF/MS or LC/MS analysis are performed after sample separation by 2D gel electrophoresis. In order to perform sensitive analysis especially by LC/MS, the high purity of the water used for preparing reagents is required. To estimate the effect of water, HPLC and LC/MS analysis were performed on commercially available bottled HPLC-grade waters and on water polished using Milli-Q water systems which have different purification technologies. As a result, UV oxidation on the purity of the ultra pure water was effective to reduce LC/MS backgrounds and noises.View full abstractDownload PDF (162K)
NOTE
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Ken'ichi KAMIJO, Hironori MIZUGUCHI, Akihisa KENMOCHI, Masao SATO, Yok ...2003 Volume 51 Issue 5 Pages 542-549
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSWe have proposed an XML format, HUP-ML (Human Proteome Markup Language), for proteomics database to exchange proteome data among researchers and to accelerate their collaboration. HUP-ML data model is proteome-analysis-oriented so as to incorporate such information as sample source, details of sample preparation, 2-D gel electrophoresis images, spot identification, amino acid sequences, MS spectrum data, and so on. We have developed prototypes of HUP-ML Editor and web-based database system as a tool for creating HUP-ML documents and a platform for sharing HUP-ML documents, respectively.View full abstractDownload PDF (364K)
COMMUNICATIONS
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Tohru YAMAGAKI, Ken-ichi HAMAYASU, Hitoshi HASHIMOTO, Hiroshi NAKANISH ...2003 Volume 51 Issue 5 Pages 550-553
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSNovel heterogeneous cyclodextrin (CyD) compounds of 6-O-(4-O-α-D-glucuronyl-α-D-glucosyl)cyclomaltoheptaose (GlcU-Glc-βCyD) and 6-O-(N-acetyl-β-D-glucosaminyl)cyclomaltoheptaose (GlcNAc-βCyD) were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Since the branch sugar residues of these heterogeneous CyDs had different molecular weights from those of glucose residues, the product ions generated from the branch or CyD part could be distinguished by their PSD spectra. In the PSD spectrum of GlcU-Glc-βCyD, the product ions, [M - Glc3~5 + Na] + , [M - GlcU + Na] + , and [M - GlcU - Glc1~5 + Na] +, were observed. The chemical species of [M - Glc3~5 + Na] + was generated by the two-site cleavage of the glycosyl linkages in the CyD part without cleavage of the branch. In the PSD spectrum of GlcNAc-βCD, [M - Glc1~5 + Na] + , and [M - GlcNAc - Glc1~4 + Na] + ions were observed.View full abstractDownload PDF (153K) -
Masatoshi NAKAGAWA, Tohru YAMAGAKI, Hiroshi NAKANISHI2003 Volume 51 Issue 5 Pages 554-558
Published: 2003
Released on J-STAGE: October 16, 2007
JOURNAL FREE ACCESSIn the sequence analyses of antigen peptides from nucleoprotein in influenza virus, it was very difficult to obtain the sufficient numbers of product ions using the post-source decay (PSD) method in MALDI-TOF-MS. Fluorescent modification of the thiol group of the cysteine residue in the target peptides using 5-iodoacetamide fluorescein was introduced for the PSD measurement. The fluorescently-labeled peptides gave sufficient product ions for complete sequence analyses in the PSD spectrum, which leads to the complete sequence analyses of the peptides with cysteine residue.View full abstractDownload PDF (166K)
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