Introduction
阮雪芬
National Taipei University of Technology
Feb 24, 2003
Outline
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Introduction to proteomics
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Definitions of Proteomics
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The major techniques in current
proteomics
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Protein-protein interaction
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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
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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
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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
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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?
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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
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Two-dimensional electrophoresis
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Mass Spectrometry
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IEF strip separation
SDS-PAGE gel separation
Protein sequencing
Peptide mapping
Others
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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.expas
ych/tools/peptide
nt.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
SDS-PAGE
(based on molecular weight)
High
MW
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
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Protein Identification by
MALDI-TOF
1. Cut protein spot
2. Protein digestion
Protease
4. Spot onto MALDI chip
5. MALDI-TOF analysis
3. Peptide purification
6. Peptide fragment fingerprint
How Does a Mass Spectrometer
Work?
Sample
input
Ionization
Analyzer
Detector
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
Ionization
Matrix-Assisted Laser Desorption/Ionization
(MALDI)
Matrix:
- organic acids
- benzoic acids
Isotope-coded Affinity Tags
Linker
(ICAT)
Biotin
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
Thiolreactive
end group
A strategy for mass spectrometric
identification of proteins and posttranslational 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
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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
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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
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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.
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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
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Body fluids
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Blood cell
Plasma and serum
Urine
Cerebrospinal fluid
Amniotic fluid
Synovial fluid
Saliva
Sweat
Tears
Semen
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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
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The Study of Protein-protein
Interaction by Mass
Spectrometry
bait
?
?
S14
?
?
SDS- PAGE
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MASS
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Yeast Two-hybrid System
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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 DNAbinding
GAL4 DNAactivation
domain
domain
Nature, 2000
Yeast Two-hybrid System
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Library-based yeast
two-hybrid
screening method
Nature, 2000
Protein-protein Interactions on
the Web
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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
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C. Elegans
http://cancerbiology.dfci.harvard.edu/cancerbiology/ResLabs/Vidal/
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H. Pylori
http://pim/hybrigenics.com
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Drosophila
http://gifts.univ-mrs.fr/FlyNets/Flynets_home_page.html
Yeast Protein Linkage Map Data
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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
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/cgibin/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 searching
FASTA Search
Programs
http://fasta.bioch.virginia.edu/
Protein and nucleotide database
searching
Cleaved
Radioactivity of
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



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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
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