蛋白質體學 阮雪芬 Jul 18 & 25, 2003. Outline The characters of proteins Differences...

蛋白質體學

阮雪芬Jul 18 & 25, 2003

Outline The characters of proteins

Differences between protein chemistry & proteomics

Why to study proteome

Proteomics Introduction to proteomics Definitions of proteomics The major techniques in current proteomics Protein-protein interactions

The characters of proteins

DNA 和蛋白質合成的地方

Three Developments Formed the Foundation of the New Biology

The growth of gene, expressed sequence tag (EST), and protein-sequence databases during the 1990s.

The introduction of user-friendly, browser-based bioinformatics tools.

The development of oligonucleotide microarray.

Why to study proteome ?

Why the Transcriptomic Analyses May Not Have Revealed All Proteins ?

Lack of correlation between transcript and disease-associated protein levels

Translocation of a protein in the disease state rather than simply differential levels of the transcript

Novel/uncharacterized genes that are not highly represented within the "closed system" of a cDNA array

Individual proteins

Complete sequence analysis

Emphasis on structure and function

Structural biology

Complex mixtures Partial sequence

analysis Emphasis on

identification by database matching

Systems biology

Protein chemistry Proteomics

Introduction To Proteomics

DNA

mRNA

Proteins

Cell functions

Genome “Genomic

s”

Proteome“Proteomics”

Genomics vs. Proteomics

Generalized Proteomics Scheme

Yarmush & Jayaraman, 2002

Definitions of 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.

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

Definitions of Proteomics

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.

Jürgen Drews, 2000

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： 698.4 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.

The Major Techniques in Current 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

3.5 1010

42

6070

150

kDa

pH

Increase of 50%

Decrease of 50%

Unmatched spots

Matched spots

Image Matching

www.expasy.ch/ch2d

http://www.expasy.ch/melanie/

Standard Proteome Analysis by 2DE-MS

Current Opinion in Chemical Biology 2000, 4:489–494

Mass Fingerprint Searching in http://www.expasych/tools/peptident.html

Yarmush & Jayaraman, 2002

Typical mass spectrometry scheme

peptide mass mapping and tandem mass spectrometry

Ionization State as a Function of pH

First dimension: IEF (based on isoelectric point)

SD

S-P

AG

E(based on m

olecular w

eight)

+ -

acidic basic

HighMW

Low MW

Sample

Two-dimensional Gel Electrophoresis

Silver staining Coomassie blue staining Sypro Ruby staining

Staining of Polyacrylamide Gels

Image Analysis

In-gel Digestion Enzyme:

trypsin chymotrypsin

*

*

**

**

*

Trypsin

Peptide mass fingerprinting (PMF) or peptide mapping

Mass Spectrometric Identification of Proteins Mapping

1. Cut protein spot 2. Protein digestion

3. Peptide purification4. Spot onto MALDI chip

5. MALDI-TOF analysis 6. Peptide fragment fingerprint

Protease

Protein Identification by MALDI-TOF

IonizationIonization

Sample Sample inputinput AnalyzerAnalyzer DetectorDetector

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(TOF), ion trap etc.

• Detection:

How Does a Mass Spectrometer Work?

Electrospray

Ionization

Matrix-Assisted Laser Desorption/Ionization (MALDI)

Ionization

Matrix:Matrix:

- organic acids- organic acids

- benzoic acids- benzoic acids

Isotope-coded Affinity Tags (ICAT)

Avidin chromatography

Biotin

Linker

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

NATURE, VOL 405, 15 JUNE 2000

A Strategy for Mass Spectrometric Identification of Proteins and Post-translational Modifications

‘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 )

Proteome chip

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 anal

ysis 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

Protein-protein Interactions

Protein-protein Interactions 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 Interactions by Mass Spectrometry

bait

S14

??

??

* *

**

SDS- PAGE

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-binding

domain

GAL4 DNA-activation domain

Nature, 2000

Yeast Two-hybrid System

Nature, 2000

Library-based yeast two-hybrid screening method

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

Pathway SoftwareBIOCARTA

http://biocarta.com/

Browse all pathway

Pathway SoftwareBIOCARTA

Pathway Result 1:Enolase Glycolysis

Pyruvate

Acetyl-CoAethanol lactate

Cancer cells

BIOCARTA

Pathway Result 2:Retinoic Acid Receptor RXR-alpha

BIOCARTA

Useful BioWebSite 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

Peptide mass mapping and sequencing

PeptIdent http://www.expasy.ch/tools/peptident. Peptide mass mapping and sequencing

PepSea http://195.41.108.38/PepSeaIntro.html

Peptide mass mapping and sequencing

MASCOT http://www.matrixscience.com/ Peptide mass mapping and sequencing

PepFrag http://www.proteometrics.com/ Peptide mass mapping and sequencing

Protein Prospector

http://prospector.ucsf.edu/ Peptide mass mapping and sequencing

FindMod http://www.expasy.ch/tools/findmod/ Posttranslational modification

SEAQUEST http://fields.scripps.edu/sequest/ Uninterpreted MS/MS searchingFASTA Search Programs

http://fasta.bioch.virginia.edu/ Protein and nucleotide database searching

Cleaved Radioactivity ofPhosphopeptides

http://fasta.bioch.virginia.edu/crp Protein phosphorylation site mapping

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)

Major Directions in Coming Proteomics

Protein structure prediction and modeling

Assignment of protein structure to genomes

Classifications of protein structures Drug discovery and development

Types of Proteomics and Their Applications to Biology

Proteomics Network

Identify Proteins

Drug Discovery

Structures

Protein-Protein Interactions

Pathways

Protein Functions

Thank You!