蛋白質體學
阮雪芬
Jul 18 & 25, 2003
Outline
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The characters of proteins
Differences between protein chemistry &
proteomics
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Why to study proteome
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Proteomics
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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 interactions
The characters of
proteins
DNA 和蛋白質合成的地方
Three Developments Formed the
Foundation of the New Biology
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The growth of gene, expressed
sequence tag (EST), and proteinsequence 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 ?
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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
Protein chemistry
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Individual proteins
Complete sequence
analysis
Emphasis on
structure and
function
Structural biology
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Proteomics
Complex mixtures
Partial sequence
analysis
Emphasis on
identification by
database matching
Systems biology
Introduction To Proteomics
Genomics vs. Proteomics
DNA
Genome
“Genomics”
mRNA
Proteins
Cell functions
Proteome
“Proteomics”
Generalized Proteomics Scheme
Yarmush & Jayaraman, 2002
Definitions of 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
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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.
Jürgen Drews, 2000
Amgen（Applied Molecular Genetics）
成立日期：1980 年4 月8 日
CEO：Kevin W. Sharer
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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
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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
kDa
150
Image Matching
Increase
of 50%
70
60
Decrease
of 50%
42
Unmatched
spots
Matched
spots
10
pH
3.5
10
www.expasy.ch/ch2d
http://www.expasy.ch/melanie/
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
Typical mass spectrometry
scheme
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peptide mass
mapping and
tandem mass
spectrometry
Yarmush & Jayaraman, 2002
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
In-gel Digestion
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Enzyme:
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trypsin
chymotrypsin
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(TOF), ion trap etc.
• Detection:
Ionization
Electrospray
Ionization
Matrix-Assisted Laser Desorption/Ionization (MALDI)
Matrix:
- organic acids
- benzoic acids
Isotope-coded Affinity Tags
(ICAT)
Linker
ICAT consists of a biotin
affinity group, a linker region
that can incorporate heavy or
light atoms , and a thiolreactive end group for linkage
to cysteines
Avidin chromatography
Biotin
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 posttranslational 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
Protein-protein Interactions
Protein-protein Interactions
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Introduction
Mass Spectrometry
Yeast Two-hybrid Assay
Introduction
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Protein-protein interactions are
intrinsic to every cellular process.
Form the basis of phenomena
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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
*
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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
domain
GAL4 DNAactivation
domain
Nature, 2000
Yeast Two-hybrid System
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Library-based yeast twohybrid 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
Pathway Software
BIOCARTA
http://biocarta.com/
Browse all pathway
Pathway Software
BIOCARTA
Pathway Result 1:
Enolase
Pyruvate
Cancer cells
Acetyl-CoA ethanol
lactate
Glycolysis
Pathway Result 2:
Retinoic Acid Receptor RXR-alpha
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
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)
Major Directions in Coming
Proteomics
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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
Protein-Protein Interactions
Protein Functions
Pathways
Drug Discovery
Structures
Thank You!