Yuki Juan ( 阮雪芬 ) Aug 7, 2003 Bioinformatics Data Analysis in Proteomics

Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

Proteomics Network Identify Proteins Drug Discovery Structures Protein-Protein Interactions Pathways Protein Functions

Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

What Is Proteomes ?

Proteomes Gene + Chromosome  Genome Protein +Genome  Proteome Proteomes are dynamics Proteome changes as a function of: time development extracellular condition intracellular condition

Definitions of Proteomics First coined in 1995 by Wilkins Be defined as the large-scale characterization of the entire protein complement of a cell line, tissue, or organism. The study of proteomes Goal: -To obtain a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually.

Proteomics Origins In 1975, the introduction of the 2D gel by O’Farrell who began mapping proteins from E. coli. The first major technology to emerge for the identification of proteins was the sequencing of proteins by Edman degradation  picomole MS technology has replaced Edman degradation to identify proteins  femtomole

How Proteomics Can Help Drug Development

Why is Proteomics Necessary? Having complete sequences of genome is not sufficient to elucidate biological function. A cell is normally dependent upon multitude of metabolic and regulatory pathways for its survival Modifications of proteins can be determined only by proteomic methodologies It is necessary to determine the protein expression level The localization of gene products can be determined experimentally Protein-protein interactions Proteins are direct drug targets.

Types of Proteomics and Their Applications to Biology

Mechanisms by Which a Single Gene Can Give Rise to Multiple Gene Products In bacteria, 1 or 2 proteins/gene In yeast, 3 proteins/gene In human, 3 or more proteins/gene Glycosylation

Two-dimensional Gel Approach Nature 2000, 405,

kDa pH Increase of 50% Decrease of 50% Unmatched spots Matched spots Image Matching

Standard Proteome Analysis by 2DE-MS Current Opinion in Chemical Biology 2000, 4:489–494 Mass Fingerprint Searching in y.ch/tools/peptide nt.html

ControlTreated sample Example: Gel

+ - gel membrane Transfer proteins from gel to PVDF membrane Sequencing (Very time-Consuming)

Mass spectrometry

For peptide mass fingerprint data

Species pI and MW

Peptide Mass Fingerprinting

World Wide Web Tools for Searching Databases Site nameURLInformation available MOWSE mass mapping and sequencing ProFoundhttp://prowl.rockefeller.edu/cgi- bin/ProFound Peptide mass mapping and sequencing PeptIdenthttp:// mass mapping and sequencing PepSeahttp:// /PepSeaIntro.htmlPeptide mass mapping and sequencing MASCOThttp:// mass mapping and sequencing PepFraghttp:// mass mapping and sequencing Protein Prospectorhttp://prospector.ucsf.edu/Peptide mass mapping and sequencing FindModhttp:// modification SEAQUESThttp://fields.scripps.edu/sequest/Uninterpreted MS/MS searching FASTA Search Programs and nucleotide database searching Cleaved Radioactivity of Phosphopeptides phosphorylation site mapping

The Information Stored in Genes Is Expressed by a Multistage Process

DNA 和蛋白質合成的地方 DNA Proteins Sugar Chain

Post-translational Modification Genomics provides comprehensive data-bases of sequence information, DNA and mRNA provide no information concerning the activities and post- translational modifications of proteins. The number of documented protein co- and post- translational modifications now exceeds 400 (http: / / abrf.org / index-.cfm/dm.home). The elucidation of protein post-translational modifications is perhaps the most important justification for proteomics as a scientific endeavor.

Chemical modification Covalent linkage of chemical group to amino acid in protein Several types: Acetylation (common on first amino acid) Phosphorylation (often involved in regulation) Lipidation (attachment to membrane) Glycosylation (common outside cell)

Processing Removal of part of protein by either: Cleavage (protease cuts protein into smaller pieces) Splicing (self-removal and rejoining of ends)

Post-translational Modification

Glycosylation Mass spectrometric “sequencing” of oligosaccharides Increase the complexity of the proteome

Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

Protein Functions Gene Ontology GoMiner

Gene Ontology

GoMiner

Genome Biology 2003, 4:R28

Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

Protein-protein Interactions Introduction Mass Spectrometry Yeast Two-hybrid Assay

Introduction Kanehisa (2000)said: Interaction NetworkFunction Post-genome informatics (2000)

Introduction Protein-protein interactions are intrinsic to every cellular process. Form the basis of phenomena DNA replication and transcription Metabolism Signal transduction Cell cycle control Secretion

Introduction Knowledge of interacting proteins Provide insight into the function of important genes Elucidates relevant pathways Facilitates the identification of potential drug targets Use in developing novel therapeutics

Interaction Discovery Methods TiBS(2003), 27(12),

Structural Biology of Interactions and Complexes X-ray crystallography Nuclear Magnetic Resonance (NMR) Electron microscopy (EI)

Examples of Three-dimensional Protein Interactions and Complexes TiBS(2003), 27(12),

The Study of Protein-protein Interaction by Mass Spectrometry bait S14 ? ? ? ? ** ** SDS- PAGE MASS

Peptide Mass Fingerprinting

Detecting Protein-protein Interactions by Protein Microarray

Yeast Two-hybrid System Useful in the study of various interactions The technology was originally developed during the late 1980's in the laboratory Dr. Stanley Fields (see Fields and Song, 1989, Nature).

Yeast Two-hybrid Assay GAL4 DNA- binding domain GAL4 DNA- activation domain Nature, 2000

Yeast Two-hybrid Assay Library-based yeast two-hybrid screening method Nature, 2000

Information Extraction (IE) A vast amount of data on protein- protein interactions residues in the published literature, which never been entered into databases. IE have been applied to gaining information on protein-protein interactions.

Mining Literature for Protein-protein Interactions

Protein-protein Interactions on the Web Yeast C. Elegans al/ H. Pylori Drosophila

Yeast Protein Linkage Map Data New protein-protein interactions in yeast Stanley Fields Lab List of interactions with links to YPD

Yeast Protein Linkage Map Data

GeneScape PathwayCalling: Protein interaction and pathway Analysis PATHCALLING YEAST DATABASE

GeneScape

Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

Biological Pathways

Metabolic and biochemical Transcription, regulation and protein synthesis Signal transduction

Metabolic and Biochemical Pathway The synthesis of fatty acids

Transcription, Regulation and Protein Synthesis Transcription Initiation Elongation Termination

Initiation of Transcription

The Elongation of Transcrption

The Termination of Transcription

Transcription, Regulation and Protein Synthesis Translation Initiation Elongation Termination

30S Subunit and Shine-Dalgarno Sequence IF: Initiating protein factor E: Exit site P: Peptide site A: Aminoacyl site

Initiation of Translation in E. coli

The Elongation Process in Protein Synthesis EF-Tu: Elongation Factor Peptide transferase

Termination of Protein Synthesis Stop codon: UAG, UAA, UGA Cytoplasmic protein release factor

Signal Transduction EDF signaling pathway Protein kinase

Pathway Conservation

Databases of Pathways---KEGG

Glycolysis

Pathway Software BIOCARTA Browse all pathway

Pathway Software BIOCARTA

Pathway Result 1: Enolase Glycolysis Pyruvate Acetyl-CoA ethanol lactate Cancer cells BIOCARTA

Pathway Result 2 : Retinoic Acid Receptor RXR-alpha BIOCARTA

Development of the Network- based Pathway Paradigm Ti BS (2003), 28(5),

Development of the Network- based Pathway Paradigm Ti BS (2003), 28(5),

BIND

Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

Protein Structure Data Bank- PDB

Therapeutic Target Classes

Amgen （ Applied Molecular Genetics ） 成立日期： 1980 年 4 月 8 日 CEO ： Kevin W. Sharer 員工人數： 6342 市場總值： 億美元 產品項目：重組蛋白藥物 EPOGENR (Epoetin alfa) NEUPOGENR (Filgrastim) INFERGENR (Interferon alfacon-1) 資料來源：彭博資訊社、 Zacks.com ， 6/14/2001

各項產品營業收入 資料來源： Amgen, Inc.

Examples of Structure-based Drug Design HIV protease with the inhibitor amprenavir (Agenerase) bound Close-up of zanamivir (Relenza) bound to influenza neuraminidase

Outline Introduction to proteomics Protein Functions Protein-protein interactions Pathways Protein Structures and Drug Discovery Bioinformatics on the Web

Useful Bio Websites PROSITE Amos' WWW links page Phosphoprotein Database (PPDB) lmmb.ncifcrf.gov/phosphoDB/

PROSITE is a database of protein families and domains.

Proteomics Network Identify Proteins Drug Discovery Structures Protein-Protein Interactions Pathways Protein Functions