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Project Outline
(1) Establishment for methods for extraction, separation and structural determination of endogenous peptides (Scheme)
1. Establishment for methods for extraction, separation and structural determination of endogenous peptides
In order to establish the methods required for constructing the fact database for endogenous peptides, we will develop
  1. the optimal extraction procedures for the peptides in the tissue and cells by minimizing the degradation of peptides and proteins,
  2. the efficient and quick condensation procedures for the peptide fraction of Mr less than 6,000,
  3. the normalized 2-dimensional high performance liquid chromatography (2D-HPLC) for the separation of the peptide fraction,
  4. the detection and structural analysis methods for the peptides mainly using mass spectrometers, and
  5. the data acquisition system for the physicochemical properties (hydrophobicity, positive charges, and molecular mass) of the peptides during the separation, detection and analysis steps.
These procedures and systems should be normalized, automated and highly sensitive to obtain the data enough for the Peptidome database.
(Invetigator)
2. High-sensitivity structural analysis of biological peptides by mass spectrometry
Mass spectrometric (MS) methods and relating techniques for high-sensitivity and high-throughput analysis of biological peptides are under study and the following results have been obtained:
  1. MS/MS sequencing of peptides using picomole level of samples,
  2. High-throughput N-terminal sequencing (up to three amino acid residues) by combination of gas-phase chemical reaction and matrix-assisted laser desorption ionization MS
  3. Development of a stable isotope-labeling method for "Peptidomics",
  4. Identification of several MS/MS fragment ions characteristic for the modified peptides such as those containing methionine sulfoxide, phosphorylated amino acid and beta-aspartic acid,
  5. Improvement of software aid for peptide sequencing based on MS/MS (http://www.protein.osaka-u.ac.jp/organic/), and
  6. Development of a search engine based on MS data to aid peptide identification from sequence databases by MS.
(Invetigator)
(2) Biological activities and structure of endogenous peptides and receptors (Scheme)
1. Highly efficient discovery system for biologically active peptides using cultured cells and orphan G protein-coupled receptors
This sub-theme is aimed to develop the efficient, systematic and highly sensitive methods that are required for screening and identifying new biologically active peptides. To detect even weak biological activity in the screening, we will set up the high throughput and sensitive system for measurements of the second messenger concentrations (intracellular calcium and cAMP) in the cultured cells as well as in the cells expressing orphan G-protein coupled receptors. By using these systems, we will search for new biologically active peptides in the extracts of porcine and rat tissue extracts, and analyze their functions when the peptides are identified. During the screening steps, we will also comprehensively collect the screening data and store them on the common platform (normalized 2D-HPLC) of the Peptidome database.
(Invetigator)
2. Isolation and characterization of bioactive peptides using functional nd developmental assay systems
Biologically active peptides interact with functional proteins (e.g. receptors, ion channels, enzymes etc.), and they are critically involved in controlling cellular signal transduction, metabolism and development. To unveil such peptides, we developed a sensitive and high-throughput screening system using cells expressing specific functional protein or cells of potential development. Molecular and physiological information of the peptides identified using the system is to be deposited to the peptide database of this project.
(Invetigator)
3. Sensitive and high throughput ligand-screening system of G protein-coupled receptors.
The ligand-screening system for G protein-coupled receptors will be developed and used for identification of peptides as endogenous ligands for orphan G protein-coupled receptors.
(Invetigator)
4. Structural modeling of peptides and peptide receptors for drug design
This project focuses on elucidation of the receptor-bound structures of bioactive peptides. We apply molecular dynamics simulations and NMR analysis to elucidation of the solution structures of bioactive peptides. Modeling of the “active form” of peptide receptors from the rhodopsin crystal structure is also a major interest in modeling the receptor-bound structures of the bioactive peptides. Those structural data will be used for designing new peptide analogs and nonpeptide molecules.
(Investigator)
(3) Construction of PEPTIDOME database (Scheme)
1-1. Research on the efficient collection and storage methods of various peptide information in the Peptidome database
The Peptidome database contains various kinds of information, as this is the fact database based on the results obtained by the experiments and measurements. Thus , it is essential to develop the database system suitable for collecting and storing huge amounts of experimental data obtained in this project, such as mass-spectrum and chromatographic data, natural abundance, processing, biological activity and receptors of the peptides as well as the fractions during the separation processes. Researchers should be easily able to register their results into the database and to search/utilize them. New general description system for the sequence and modified structure of peptides will also be set up for the mass spectrometric analysis and database searching. Moreover, we will also develop the system to get related information of the target peptides efficiently and quickly from the reference databases.
(Invetigator)
1-2. Construction of Peptidome database efficient for searching and discovery
To construct the Peptidome database that is easily searched by the researchers, we will develop the relational fact database, which systematically stores the different types and huge amounts of data based on the physiocochemical and other properties of the peptides. We further study the extensibility of the Peptidome database for the future increments of amounts and diversity of the peptide data. The data mining and discovery system will also be developed to deduce unknown activity of the peptides as well as to fill up the deleted data from the analysis and comparison of the stored peptide data by maximally utilizing the features of this database.
(Invetigator)
2. Research for relational system between biological databases.
There are hundreds of biological databases in the internet world, which is maintained independently. To make the 'Peptidome Database' useful for their users, it is important to fit the database within the network of biological databases. Linking the URL in WWW system is a useful and well known method to overcome this issue. An automated system to create this link during the update of each database, and fitting Peptidome database to this system is under development. Also, it is important to FEEL the peptide as they behave in the micro-environment. I am developing a virtual reality system with force feed back to feel the potential they have.
(Invetigator)