阮雪芬 National Taipei University of Technology Feb 24, 2003 Introduction 阮雪芬 National Taipei University of Technology Feb 24, 2003

Outline Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Major Directions in Coming Proteomics

Outline Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Major Directions in Coming Proteomics

What Is Proteomics ?

Proteomics Protein +Genome Proteome ProteomeProteomics

Outline Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Major Directions in Coming Proteomics

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

Definitions of Proteomics The classical definition Two-dimensional gels of cell lysate and annotation. Two-dimensional gels to visualize differential protein expression. In the post-genomics era Protein Identification Post-translational modifications Determining Function Molecular Medicine Differential display by two-dimensional gels Protein-Protein Interactions

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 http://www.sciam.com.tw/read/readshow.asp?FDocNo=63&CL=18

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

Outline Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Major Directions in Coming Proteomics

The Major Techniques in Current Proteomics Two-dimensional electrophoresis IEF strip separation SDS-PAGE gel separation Mass Spectrometry Protein sequencing Peptide mapping Others ICAT Yeast two hybrid assay Protein chips

Two-dimensional Gel Approach Nature 2000, 405, 837-846

Standard Proteome Analysis by 2DE-MS Mass Fingerprint Searching in http://www.expasych/tools/peptident.html Current Opinion in Chemical Biology 2000, 4:489–494

Ionization State as a Function of pH

Two-dimensional Gel Electrophoresis First dimension: IEF (based on isoelectric point) + - Sample acidic basic High MW (based on molecular weight) SDS-PAGE Low MW

Staining of Polyacrylamide Gels Silver staining Coomassie blue staining Sypro Ruby staining

Image Analysis

Mass Spectrometric Identification of Proteins - Mapping Peptide mass fingerprinting (PMF) or peptide mapping * Trypsin

Protein Identification by MALDI-TOF 1. Cut protein spot 2. Protein digestion 3. Peptide purification 4. Spot onto MALDI chip 5. MALDI-TOF analysis 6. Peptide fragment fingerprint Protease

How Does a Mass Spectrometer Work? Sample input Analyzer Detector Ionization

How Does a Mass Spectrometer Work? Sample Input: Gas Chromatography (GC), Liquid Chromatography (LC), Capillary Electrophoresis (CE), Solid crystal etc. Ionization: Electrospray, Matrix-assisted Laser Desorption/Ionization (MALDI) etc Analysis: quadrupole, time of flight, ion trap etc. Detection:

Ionization Electrospray

Matrix-Assisted Laser Desorption/Ionization (MALDI) Matrix: - organic acids - benzoic acids

Isotope-coded Affinity Tags (ICAT) Linker Biotin Thiol-reactive end group ICAT consists of a biotin affinity group, a linker region that can incorporate heavy or light atoms , and a thiol-reactive end group for linkage to cysteines Avidin chromatography

A strategy for mass spectrometric identification of proteins and post-translational modifications NATURE, VOL 405, 15 JUNE 2000

Proteome chip ‘proteome chip’ composed of 6,566 protein samples representing 5,800 unique proteins, which are spotted in duplicate on a single nickelcoated glass microscope slide39. The immobilized GST fusion proteins were detected using a labeled antibody against GST. (MacBeath G. Nat Genet 2002 Dec;32 Suppl 2:526-32 )

Microarrays for Genomics and Proteomics DNA microarray are used for genetic analysis as well as expression analysis at the mRNA level. Protein microarrays are used for expression analysis at the protein level and in the expansive field of interaction analysis.

Protein Microarrays In Medical Research Accelerate immune diagnostics. The reduction of sample volume----the analysis of multiple tumor markers from a minimun amount of biopsy material. New possibilities for patient monitoring during disease treatment and therapy will be develpoed based on this emerging technology.

Clinical and Biomedical Applications of Proteomics An approach complementary to genomics is required in clinical situations to better understand epigenetic regulation and get closer to a "holisitic" medical approach. The potential clinical applications of 2-D PAGE, especially to the analysis of body fluids and tissue biopsies. Identifying the origin of body fluid samples or the origin of a tissue biopsy. Analyzing protein phenotypes and protein post-translational modifications in fluid, cells, or tissues. Examining the clonality of immunoglobulins and detecting clones which are not seen with conventional techniques. Monitoring disease processes and protein expression. Discovering new disease markers and/or patterns in body fluids, cells, or tissues.

Clinical applications of 2-D electrophoresis Body fluids Blood cell Plasma and serum Urine Cerebrospinal fluid Amniotic fluid Synovial fluid Saliva Sweat Tears Semen Solid tissue Heart Brain Thyroid Muscle Malignant diseases Tissue culture Malignant cells Bacterial proteins Young & Tracy Journal of Chromatography A, 698 (1995) 163-179

Outline Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Major Directions in Coming Proteomics

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

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

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

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 System GAL4 DNA-activation domain GAL4 DNA-binding domain Nature, 2000

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

Protein-protein Interactions on the Web Yeast http://depts.washington.edu/sfields/yplm/data/index.html http://portal.curagen.com http://mips.gsf.de/proj/yeast/CYGD/interaction/ http://www.pnas.org/cgi/content/full/97/3/1143/DC1 http://dip.doe-mbi.ucla.edu/ http://genome.c.kanazawa-u.ac.jp/Y2H C. Elegans http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/Vidal/ H. Pylori http://pim/hybrigenics.com Drosophila http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html

Yeast Protein Linkage Map Data New protein-protein interactions in yeast List of interactions with links to YPD Stanley Fields Lab http://depts.washington.edu/sfields/yplm/data

Yeast Protein Linkage Map Data

Information available Useful BioWeb Site name URL Information available MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse Peptide mass mapping and sequencing ProFound http://prowl.rockefeller.edu/cgi-bin/ProFound PeptIdent http://www.expasy.ch/tools/peptident. PepSea http://195.41.108.38/PepSeaIntro.html MASCOT http://www.matrixscience.com/ PepFrag http://www.proteometrics.com/ Protein Prospector http://prospector.ucsf.edu/ FindMod http://www.expasy.ch/tools/findmod/ Posttranslational modification SEAQUEST http://fields.scripps.edu/sequest/ Uninterpreted MS/MS searching FASTA Search Programs http://fasta.bioch.virginia.edu/ Protein and nucleotide database searching Cleaved Radioactivity of Phosphopeptides http://fasta.bioch.virginia.edu/crp Protein phosphorylation site mapping

Outline Introduction to proteomics Definitions of Proteomics The major techniques in current proteomics Protein-protein interaction Major Directions in Coming Proteomics

Major Directions in Coming Proteomics Chemical proteomics (screens for activity and binding) Structural proteomics (target validation and development) Interaction proteomics (identification of new protein targets) Bioinformatics (annotation of the proteome)

Bioinformatics in Coming Proteomics Protein structure prediction and modeling Assignment of protein structure to genomes Drug discovery and development