Introduction of Proteomics

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Introduction of Proteomics
Shui-Tein Chen Institute of
Biological Chemistry Academia
Sinica. Taiwan
12-10-04
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• Outline
• Introduction of proteomics
• 2. Proteomics and Systems Biology.
• 3. Diagnostic and therapeutic.
• 4. Protein markers and disease
• 5. Advanced Technology for Proteomics study
• I-CAT, Aptamer, I.P. multi-dimentional C.E. etc.

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What is a Proteome?
Proteome PROTEin complement expressed by a
genOME or tissue (Wilkins et al., 1995
Biotechnology and Genetic Engineering Reviews 13,
19-50.)
Proteomes are dynamic
Proteomes change as a function of - time
- development - extracellular
conditions - intracellular conditions
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What is Proteomics ?
-proteomics is the study of proteomes
-proteomics aims to -separate identity and
characterize proteins on a large scale
-define levels of proteins in cells / tissues and
how these change -investigate protein
complexes -elucidate protein functions,
pathways, and interrelationships
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Proteomics Proteomics analysis is the analysis of
the PROTEin complement expressed by a genOME.
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• How can you separate and visualise the proteins
  in a proteome?
• how can this be used to study protein complexes
  and pathways?
• how can you quickly identify separated proteins?
• -how can you characterise protein in detail?
• -why do companies want to spend millions on
  proteomics?

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Traditional Protein Chemistry

• Column chromatography
• (e.g. ion exchange or reversed phase)
• one protein at a time
• purification may take months

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Proteomics Array Two dimensional gel
electrophoresis

• First dimension charge-base
• Separation.
• Second dimention mass-based separation.
• Up to thousands of proteins purified at once.
• Proteins purified in parallel in
• 1-3 days.
• Image is reference map for cell, tissue or
  protein complex.

3 Protein pI 10
200KD
Protein Mass
10kd
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The techniques availableNorthern blots (for gene
expression) and Western blots (for protein
levels) made charting the status of more than a
handful of genes or proteins a formidable
analytical task.
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Proteomics and the New Biology The
New Biology Proteomics is the study of the
proteome, the protein complement of the genome.
proteomics ? proteome genomics ?
genome Until the mid-1990s, biochemist,
molecular biologists, and cell biologists studied
individual genes and proteins or small clusters
of related components of specific biochemical
pathways.
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Proteomics? Thats just what we used to call
protein chemistry. Differences between
protein chemistry and proteomics -----------------
--------------------------------------------------
--------------------- Protein chemistry

Proteomics ---------------------------------------
------------------------------------------------ I
ndividual proteins
complex mixtures Complete sequence analysis
partial sequence
analysis Emphasis on structure and function
emphasis on identification

by database matching Structural biology
systems biology
-------------------------------------------------
--------------------------------------- Using
physical biochemistry or mechanistic enzymology,
study one protein or multisubunit protein complex
at a time. Study multiprotein systems, in which
the focus is on the interplay of multiple,
distinct proteins in their roles as part of a
larger system or network. The point of proteomics
is to characterize the behavior of the system
rather than the behavior of any single component.
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Traditional analysis vs systems biology
http//www.sciencemag.org/cgi/content/full/291/550
7/1221/F1
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• Three developments changed the biological
• landscape and formed the foundation of the
• new biology.
• The growth of gene, expressed sequence tag (EST),
  and protein-sequence databases during the 1990s.
• ---The genome-sequencing projects of the
  late 1990s yielded complete genome sequences of
  bacteria, yeast, nematodes, and drosophila, plant
  genomes and human genome.
  
• ---These genome-sequence databases are the
  catalogs from which much of our understanding of
  living systems eventually will be extracted.
  

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• 2. The introduction of user-friendly,
  brower-based bioinformatics tools to extract
  information from these databases.
• It is now possible to search entire genomes
• for specific nucleic acid or protein sequences
• in seconds.
• This array of free web-based tools now enables
• the biologist to probe structures and functions
• of genes and gene products and to explore a great
  
• deal of interesting biochemistry right
• from a desktop computer.
  

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3. The DNA array. One array can replace
thousands of Northern-blot analyses and can be
done in the time it would take to do one
northern. Moreover, with two-color
fluorescent probe labeling, expression of genes
in two different samples can be compared directly
on one slide or chip.
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Microarray Data 1. From 6033 human genes,
approximately 5950 (98.6) of the genes were
expressed in HL-60 cell. 2. A total of 624 genes
(10.5) were found to be regulated during HL-60
cell differentiation. 3. Most of these genes
were not previously associated with HL-60 cell
and included genes encoding secreted proteins as
well as genes involved in cell adhesion,
signaling.
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• From a single array, one can assess the
  expression of all genes in the cell genome.
• We can see the whole system, but the information
  contained in these thousands of data points is
  beyond our ability to interpret intuitively.
• New clustering algrithms, self-organizing maps,
  and similar tools represent the latest approaches
  to rendering the data in ways that biologists can
  comprehend.

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The most important thing about arrays in this
context is that they have challenged biologists
to think big. A cell has thousands or tens of
thousands of genes that may be expressed in
varying combinations. The life and death of
cells is dicated by the expression of these
genes and the activities of their protein
products.
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• Each protein, whether a chaperone, expresses a
  function that assumes significance only in the
  context of all the other functions and activities
  also being expressed in the same cell.
• Thus biologist are now struggling
• to think big, to understand systems rather than
  just components, and
• to make sense of complexity.

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If we can measure gene expression, why bother
with proteomics? DNA array offer a snapshot of
the expression of many or all genes in a cell.
Unfortunately, mRNA levels tell us nothing
about the regulatory status of the corresponding
proteins, Whose activities and functions are
subject to many endogenous posttranslational
modifications and other modifications by
environmental agents.
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Characterizing Proteins and DNA at the Molecular
Level is the Key to Understanding their Function
Functional genomics
Genomics
Proteomics
2001 Proteomics Group, Institute of Biological
Chemistry, Academia Sinica
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Mass Data Collection for Protein Identification
sample
tryptic digestion
mass spectrometry (peptide mapping information)
2-D gel purification
Protein search
identification
database
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• The task of characterizing the proteome
• requires analytical methods to detect and
• quantify proteins in their modified and
• Unmodified forms.
• Tools provides the technology of proteomics.
• Database Protein, EST, and complete
  genome-sequence databases collectively provide a
  complete catalog of all proteins expressed in
  organisms for which the databases are available.
• 2. Mass spectrometry
• a, provide accurate molecular mass measurements
  of intact proteins as large as 100kDa.
• b, provide accurate mass measurement of peptides
  from proteolytic digests,
• c, provide sequence analysis of peptides
  obtained from proteolytic digests.

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3. Collection of software that can match MS data
with specific protein sequences in
databases a, take un-interpreted MS data and
match it to sequences in protein, b, survey MS
data for protein-sequence matches, c, inspect
the results and evaluate the quality of the
data. 4. Analytical protein-separation
technology, a, they simplify complex protein
mixtures by resolving them into individual
proteins or small groups of proteins, b, they
permit apparent difference in protein levels to
be compared between two samples, protein
analytical separations allow investigators
to target specific proteins for analysis.
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Proteomics analysis in the control and
experimental samples
IEF
decrease
increase
SDS-PAGE
disappear
appear
Shift
Control
Experimental
2001 Proteomics Group, Institute of Biological
Chemistry, Academia Sinica
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Platform of Proteomics
Sample preparation
2-D separation
Image analysis
A (ex. Normal)
Trypsin digestion
B (ex. Cancer)
Mass analysis
Data bank search
New protein mark
database
2001 Proteomics Group, Institute of Biological
Chemistry, Academia Sinica
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• Applications of proteomics
• Mining
• 2. Protein-expression profiling,
• 3, protein-network mapping, and
• 4, mapping of protein modifications.
• Mining identifying all of the proteins in a
  sample,
• Protein-expression profiling
• identification of proteins in a particular
  sample
• as a function of a particular state of the
  organism
• or cell or as a function of exposure to a drug,
  chemical,
• or physical stimulus.

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Protein-network mapping To determining how
proteins interact with each other in living
systems. Most proteins carry out their
functions in close association with other
proteins. Determine the functions of protein
functional networks, such as signal-transduction
cascades and complex biosynthetic or degradation
oathways. Mapping of protein modifications To
identifying how and where proteins are modified.
Many PTM govern the targeting, structure,
function, and turnover of proteins.
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Breakthrough Technology Can Transform Businesses
Examples of Technology Breakthroughs
(value creation gt100 billion)
Breakthrough
Impact
1900s Automobile Revolutionized
transportation 1920s Television Displaced radio
as key medium 1950s Research-based Enabled modem
medicine Pharmaceuticals 1980s Personal
computer Brought computing power to the
individual 1980s Cellular telephone Revolutionizin
g personal communication 1990s Internet Revoluti
onizing communication and media 2000s Biotechnolog
y ? Transformation of medicine to a
personalized and precise science Genomics Pr
oteomics New diagnostic markers and drug targets
2001 Proteomics Group, Institute of Biological
Chemistry, Academia Sinica
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Scientific American, April 2002
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Scientific American, April 2002
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Scientific American ???, 2002?4??
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Scientific American, April 2002
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Proteome Sciences (Cobham, U.K.) reported the
development of a blood assay for stroke that, the
company claim, could be the first test of its
kind used in clinical applications. The blood
markers, identified using proteomes protein
separation technologies, can diagnose stroke and
also differentiate between stroke and heart
attack. The company claims that studies by the
University Cantonal Hospital (Geneva,
Switzerland) found the assay to be 100 specific
and 68.2 sensitive, with a positive predictive
accuracy of 100.
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Proteome Sciences used high-sensitivity
protein-separation techniques to identify A
blood protein marker for stroke, which is being
developed into a blood test to enable Clinicians
to diagnose stroke and differentiate between
stroke and heart attack.
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Proteome Sciences (Cobham, U.K.) reported the
development of a blood assay for stroke that, the
company claim, could be the first test of its
kind used in clinical applications. The blood
markers, identified using proteomes protein
separation technologies, can diagnose stroke and
also differentiate between stroke and heart
attack.
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Proteomics study of Tumor markers in Nuce Mice
by Dr Chen, Jenn-Han Dr Chen, Yu-Ju
Dr Juan, Hsueh-Fen

• Nude mice---T cell-defected, so we can ignore the
  immuno-reaction by human cancer cell.
• We suppose cancer markers will be found in serum
  after tumor formation.
• Approach
• Human cancer cells ?nude mice ?serum? 2D?
  MALDI-MS or MS-MS

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MW
97
Hemopexin
66
45
30
20.1
S10
S9
14.4
pI
5
6
pI
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Cancer Markers in Different Cancer Cell line
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HUMAN
Stomach Cancer Patient
Normal Human
Control 02-0978 ???
MW
MW
SAA4
SAA1
SAA2
10
3
10
3
pI
pI
47
S3
A
B
C
D
S7
S10
E
F
S9
48
A
D
B
E
C
F
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Conclusion and Discussion

• We found some spots were different in many cancer
  cell lines and control and have identified these
  proteins.
• We found SAA are very interesting because they
  just increased in SCM1 cancer cell line.
• Using proteomic method to search cancer markers
  with nude mice model has been never reported ? we
  wish the idea and results would be prepared for
  publication.

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Cy3 Cy5 Maleimide Mono-Reactive Dye
Cy3
Cy5

• A new fluorescent labeling reagents based on
  sulfoindocyanine dyes
• Cy3 and Cy5 mono-functional maleimides are
  particularly suitable for the selective labeling
  of molecules containing free sulfhydryl groups,
  such as cysteine residues in proteins and
  peptides, oligonucletides, and antibody
• Cy3 (red) green spectrum at 552 nm , Cy5
  (green) red spectrum at 650 nm

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??King Mongkuts?? Dr. Narumon Jeyashoke Rice
Bran
Comparison of 2-D gel between two rice varieties
Rice 105
Sticky Rice
2001 Proteomics Group, Institute of Biological
Chemistry, Academia Sinica
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Gel Image
97000
66000
45000
30000
20100
14400
4.0
5.0
6.0
7.0
10.0
8.0
9.0
3.2
5.5
4.0
5.0
6.0
7.0
10.0
8.0
9.0
3.2
5.5
CNT1 labeled with Cy3
KDML105 labeled with Cy5
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Combined 2 images
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Poisonous Snakes
??? (Trimeresurus mucroquamatus)
??? (Trimeresurus stejnegeri)
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9. Ophiophagus hannah 10.
Deinagkistrodon acutus
pH 3
pH 10
pH 3
pH 10
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Isolatation and Separation of glycoproteins from
non-glycoproteins by ConA- agarose affinity
fractionation and elution by free mannose.
Protein sample 3 mg
Procedure
Disolved in Tris-HCl buffer 150 mM
NaCl 300 ml
Con A bound agarose 2 x 0.15 ml per sample
Mix, vortex
Wash with buffer 500 ml two times
centrifuge
Remove supernatant
Supernatant put in to Con A- agarose gel
Stired over night
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Con A non-binding proteins
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Multidimensional analysis of
proteome. Whole cell map of the proteome HPLC
fraction and proteomic analysis of each fractions
60 B
5 B
40.0
20.0
Retention times (min)
HPLC chromatogram
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HPLC chromatogram
min
pI 3 pI 10
pI 3 pI 10
pI 3 pI 10
pI 3 pI 10
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Albumin remove kit
???(5µl)
Albumin remove tag Unbinding
Albumin remove tag binding
94 67 45 30 20.1 14.4
94 67 45 30 20.1 14.4
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Procedure
Human serum
Molecular cut-off(10kDa)
lt10KDa proteins
gt10 KDa protein
glycoprotein removed
Albumin removed
IgG removed
Proteomic gel
Multicolor layer comparative method
Biological mass spectrometry
Target protein
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Different sections combined
?
Original proteins
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30
100
50
B? albumin bind????
B? albumin unbind?????
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Albumin bind proteins???????
???
Albumin bind
Albumin unbind proteins???????
Albumin unbind
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