proteomics and bioinformatics 阮雪芬 ntu dec 25, 2002

Proteomics and Bioinformatics

阮雪芬NTUDec 25, 2002

Outline Proteomics

Introduction to proteomics

Definitions of Proteomics

The major techniques in current proteomics

Protein-protein interaction

Bioinformatics Protein-protein Interactions

Useful Bio-websites

Major Directions in Coming Proteomics

What Is Proteomics

?

Proteomics Protein +Genome Proteome ProteomeProteomics

Outline Proteomics






Useful Bio-websites


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 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 degradationpicomole

MS technology has replaced Edman degradation to identify proteinsfemtomole

How Proteomics Can Help Drug Development

Why is Proteomics Necessary? Having complete sequences of genome is not suffic

ient to elucidate biological function. A cell is normally dependent upon multitude of me

tabolic and regulatory pathways for its survival. Modifications of proteins can be determined only b

y proteomic methodologies. It is necessary to determine the protein expression l

evel. The localization of gene products can be determine

d experimentally. Protein-protein interactions. Proteins are direct drug targets.

Types of Proteomics and Their Applications to Biology

Outline Proteomics






Useful Bio-websites


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

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

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

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

*

*

**

**

*

Trypsin

Peptide mass fingerprinting (PMF) or peptide mappingPeptide mass fingerprinting (PMF) or peptide mapping

Mass Spectrometric Identification Mass Spectrometric Identification of Proteins - Mappingof 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

IonizationIonizationSample Sample inputinput

AnalyzerAnalyzerDetectorDetector

How Does a Mass How Does a Mass Spectrometer Work?Spectrometer Work?

• Sample Input:Sample Input: Gas Chromatography (GC), Liquid Chromatography (LC), Gas Chromatography (GC), Liquid Chromatography (LC), Capillary Electrophoresis (CE), Solid crystal etc.Capillary Electrophoresis (CE), Solid crystal etc.

• Ionization: Ionization: Electrospray, Matrix-assisted Laser Desorption/Ionization Electrospray, Matrix-assisted Laser Desorption/Ionization

(MALDI) etc(MALDI) etc

• Analysis: Analysis: quadrupole, time of flight, ion trap etc.quadrupole, time of flight, ion trap etc.

• Detection: Detection:

How Does a Mass How Does a Mass Spectrometer Work?Spectrometer Work?

ElectrosprayElectrospray

IonizationIonization

Matrix-Assisted Laser Desorption/Ionization Matrix-Assisted Laser Desorption/Ionization

(MALDI)(MALDI)

Matrix:Matrix:

- organic acids- organic acids

- benzoic acids- benzoic acids

IonizationIonization

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 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 cl

inical 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 tis

sue biopsy. Analyzing protein phenotypes and protein post-translational mo

difications in fluid, cells, or tissues. Examining the clonality of immunoglobulins and detecting clon

es 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 Proteomics






Useful Bio-websites


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

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

Library-based yeast two-hybrid screening method

Nature, 2000

Outline Proteomics






Useful Bio-websites


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

Stanley Fields Lab http://depts.washington.edu/sfields/yplm/data

List of interactions with links to YPD

Yeast Protein Linkage Map Data

GeneScape PathwayCalling: Protein interaction and pat

hway Analysis

http://portal.curagen.com

PATHCALLINGYEAST DATABASE

GeneScape

GeneScapeMIPS Currently about 9750 protein-protein-interactions

(8250 physical and 1500 genetic) are annotated.

Yeast Interacting Proteins Database (YIPD)

http://genome.c.kanazawa-u.ac.jp/

Yeast Interacting Proteins Database

Genetic Network Visualization System

Workbench System for Support of Gene Regulatory Network Construction

YIPD

Java Applet

Java Applet

YIPD

GUI SystemHelp

YIPD

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


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

PepSea http://195.41.108.38/PepSeaIntro.html


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

Outline Proteomics






Useful Bio-websites



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 stability and folding Applications of hydrophobicity Superposition of structures, and structural al

ignments DALI (Distance-matrix alignment) Evolution of protein structures Classifications of protein structures

Major Directions in Coming Proteomics Protein structure prediction and modeling Assignment of protein structure to genomes Prediction of protein function orthologues a

nd paralogues Drug discovery and development

proteomics and bioinformatics 阮雪芬 ntu dec 25, 2002

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