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Dec 3, 2012 (4 years and 10 months ago)

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New Electrophoretic Separation Systems for Proteomics Studies

Y. Diana Liu and James J. Bao,

Baulo Laboratories, 6736 Hummingbird Dr., Mason, OH 45040, USA

Abstract

Genome and proteome represent some of the most astonishing revolutions in science in the
last five years.
Techniques that facilitate the studies in these fields have also gained rapid development at the same time.
For example, a key technology for proteome study, 2
-
D gel electrophoresis has been used for the
separation of thousands of protei
ns making it feasible for their identification. However, 2
-
D gel
electrophoresis has to be automated before it is possible to take advantages of the other automated
technologies, such as mass spectrometry. Several new technologies have been developed rec
ently to
enable the automation of 2
-
D electrophoresis for proteomic study and to accelerate the drug discovery
process.


Table of Contents

Abstract
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1

Introduction

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...

1

Why Proteo
mics

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2

Challenges in Proteomics

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3

Technologies for proteomics

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.....

3

2
-
D electrophoresis vs. Proteomics

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4

Limitations of Current 2
-
D electrop
horesis

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4

New Electrophoretic Systems

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5

Automation of 2
-
D Gel Electrophoresis

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5

Parallel Capillary Electrophoresis Systems

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5

Biochips

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.....

6

New Integrated 2
-
D Capillary Electrophoresis System

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7

Conclusions

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...

8

Future

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8

References

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9


Introduction

I
n the long history of science and humanity, it is often the development of new technologies that
revolutionize our view of the world as well as our lives. The invention of compass made sailing around
the world a possibility. The inventions of automobile
and computer have totally changed our life styles
today. The development of modern medicine has significantly improved the quality of our lives.
Usually, it is the development of one key technology that results in a series of new developments.
Recently,

genome has become a generic term for "big science" molecular biology. The genome projects
have captured the imagination of scientific community in the pharmaceutical and biotechnology industry
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-
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[1]. By sequencing the entire genome of an organism, here fo
r the first time in biology is the complexity
of an organism understood at the level of information content. The information content obtained from
genome study and DNA sequencing will reveal the secrets in each organism, which dictate the formation
of pro
teins. The genomes of many organisms including human have been completely sequenced and
many others are slated for completion over the next few years. While completing the sequence of each
genome represents a major scientific achievement, the utility of
the information buried within each
genetic blueprint is only valuable if it can lead to an understanding of disease
-
state processes and/or to
therapeutic targets. The challenge for us is to develop rapid methods that exploit genetic information and
lead t
o an understanding of the underlying biochemical function of gene products, thus providing new
avenue for therapeutical intervention.

The most direct way to take advantage of the known genomic information is by comparative
protein expression profiling or

proteomics. We need to define the "proteome" before we arrive at the
ultimate definition of the core structural and functional molecules, through which other molecules (e.g.
fats, carbohydrates) are synthesized in an organism. "Proteome" indicates the P
ROTEins expressed by a
genOME or tissue. Proteomics describes the practice of studying proteome. By combining separation,
visualization and quantitation of protein on 2
-
D gel electrophoresis with protein identification via in
-
gel
digestion, mass spectrom
etry and database search.

Why Proteomics

The proteome, unlike the genome, is not a fixed feature of an organism. Instead, it changes with
the state of development, the tissue or even the environmental conditions under which an organism finds
itself. There
are therefore many more proteins in a proteome than genes in a genome, especially for
eukaryotes. This is because there are many ways that a gene is spliced in constructing mRNA, and there
are many ways that the same protein can be post
-
translationally alt
ered. A means of displaying and
studying the products (proteins) of genes directly is an attractive way of studying not only disease, but
also any complex problems in biology. Molecular understanding of how a cell operates in sickness and in
health requi
res knowledge of the proteins and other cell components that are actively present, how they
interact and the outcome of their interactions. The first step in such a major task is to find what parts of
the genome are expressed, how many products are made, a
nd how the products are modified.
"Proteomics" is a direct approach, by using various technologies, to achieve the goal of identifying,
quantitating and studying the post
-
translational modifications to proteins in cells, tissues or even
organisms. Such stu
dies conducted in healthy and diseased tissues should, by deferential analysis, give
insight into what is altered. Therefore, screening proteins from cells is the ultimate solution to
understanding the disease and thus, to drug discovery.

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-
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Challenges in

Proteomics

The problem is that until very recently protein science has been a slow and frustrating art. Unlike
the development of DNA sequencing and mRNA screening, where literately thousands of genes can be
rapidly analyzed in a well
-
equipped laboratory
, the purification and characterization of a single protein
takes a lot more effort. However, the dramatic changes in proteomics that have been converging in
protein science and bioinformatics to produce the revolution that allows proteins, like DNA and m
RNA,
to be subject to mass screening.

Protein technology is inherently more complex than DNA
-
based technology. Not only is the basic
alphabet bigger (4 nucleotides for DNA vs. 20 unmodified and many more modified amino acids for
proteins), but some gene
s can be variously spliced therefore making numerously different products from a
single stretch of DNA. Additionally, mRNA editing is relatively common, leading to modified messages
and corresponding protein products. There are also many ways in which pr
oteins are modified after they
have been synthesized. It can be argued with some justification that possibly all eukaryotic proteins are
post
-
translationally modified in some way (e.g. truncation at the N
-

or C
-

terminus, by protein splicing
(rarely), or
by addition of various substituents such as sugars, phosphate, sulphate, methyl, acetyl or lipid
groups). To make matters still more complicated, while an organism has effectively a single genome (if
unusual circumstances such as the genes involved in ant
ibody production in B
-

and T
-
cells are set aside),
there are many proteomes. Even in a unicellular organism, the expressed proteins (proteome) will be
different depending on the growth conditions.

Technologies for proteomics

The technologies required to

separate large numbers of proteins in a proteome, to identify them,
and to study their modifications are by no means straightforward. As yet there is no equivalent to the
Applied Biosystem 377 high
-
throughput DNA sequencer, which has served the genome pr
ojects so well.
However, many technologies are converging and making proteome analysis possible. The most
commonly used techniques for proteomics today include sample preparation, two
-
dimensional (2
-
D) gel
electrophoresis, post separation analysis (imagi
ng, mass spectrometry), and bioinformatics (Fig 1). The
major driving force behind pursuing such a lengthy endeavor is for drug discovery.

At the heart of the technologies for proteomics is the 2
-
D gel electrophoresis. The separation of
proteins is a pre
-
condition for the identification, modification and characterization of these proteins using
the subsequent techniques. Although some non
-
electrophoretic technologies such as microbore HPLC
and flow injection mass spectrometry can also be used either alon
e or in combination with electrophoretic
technology for proteomics, 2
-
D electrophoresis is still the most dominant technology for proteome
studies.

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-
D gel electrophoresis



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2
-
D electrophoresis
vs.

Proteomics

Since the first introduction of 2
-
D electrophoresis by O’Farrell in 1995

[2], 2
-
D electrophoresis
has been used for the separation of hundreds and thousands of proteins in various forms in many
laboratories. The 2
-
D electrophoresis provides highly purified proteins that are separated in a simple
parallel process. The first d
imension of 2
-
D polyacrylamide gel electrophoresis (PAGE) is isoelectric
focusing (IEF), during which proteins are separated in a pH gradient until they reach a stationary position
where their net charge is zero. In the second dimension, the proteins sepa
rated by IEF are separated
orthogonally by electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulphate
(SDS
-
PAGE). Currently, 2
-
D gel electrophoresis is the only method available which is capable of
simultaneously separating thousan
ds of protein [3]. Using 2
-
D electrophoresis, individual proteins can be
separated, identified, and characterized though a slow and tedious process. Benefited from the massive
databases of inferred protein sequence established by DNA sequencing initiativ
es, protein identification
in the genome era is easier than it was in earlier times.

However, the emphasis has gone from a focus on protein identification to protein
characterization. Already we are not content just to know the identity of a protein.

We want to know
how much of the protein is present in the cell, and if it starts (its N
-
terminus) and ends (its C
-
terminus)
where the molecular biologists predicted it should. With eukaryotes we often find that it doesn't! Also,
we would like to know th
e mass of the protein, as this will indicate if the protein is likely to be post
-
translationally modified. If we think that a protein has modifications, we might want to further
characterize the protein and do this on a single protein spot from a 2
-
D gel.

Perhaps we would like to be
able to profile the N
-
linked sugars and finally estimate the O
-
linked sugars and finally estimate the
amounts of phosphoamino acids (phospho
-
serine,
-
threonine and
-
tyrosine). With the rapid development
in other analytical tech
niques, such as computer imaging and mass spectrometry, all of these are
becoming possible on picomole amounts of proteins. Unfortunately, 2
-
D electrophoresis as it is today is
becoming the major limiting step in the whole process of answering these quest
ions.

Limitations of Current 2
-
D electrophoresis

The most obvious and important limitation of the 2
-
D electrophoresis is quantitation. From a
practical point of view, most physiological and pathological processes are associated with quantitative
variati
on in the amounts of gene products. The most widely used techniques to detect protein spots on 2
-
D gels are silver staining and Coomassie Blue staining. None of these detection methods is very
quantitative. They are far from stoichiometric and need prote
in specific staining density vs. protein
concentration curves. They have uneven staining density at saturated spots and have low linearity ranges
(40
-
50 fold ranging from 0.04 to 2 ng/mm2 for silver staining and 20 fold ranging from 10 to 200
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-
D gel electrophoresis



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ng/mm2 for C
oomassie Blue staining). All of above difficulties with protein staining remain to be
challenges to protein quantitation. These challenges must be solved before 2
-
D polyacrylamide gel
electrophoresis (PAGE) databases can be extended to consider protein a
bundance as components of
pathological responses.

The second limitation of the current 2
-
D gel electrophoresis is its interface with other
technologies. The current electrophoresis takes several hours and even days to complete and has to be
done in separa
te steps. The results have to be examined visually first and comparisons are made manually
before a decision can be made on which protein should be sequenced. This makes it very difficult for
other instruments, such as MS, to be interfaced with this inst
rument.

Another limitation of the current 2
-
D PAGE is its complexity. In the eyes of many researchers
not "in the field", 2
-
D PAGE is still yet to become a routine separation vehicle.

New Electrophoretic Systems


Facing the challenges, scientists hav
e been working hard towards simplifying the interface
between the IPG and the SDS
-
PACE as well as increasing the high throughput of the 2
-
D PAGE [3].
Actually, an intense race to improve proteomics tools will develop over the next few years, according to

a
report released by Cambridge Healthtech Institute, Newton, Massachusetts, USA (617
-
630
-
1300), entitled
“Proteomics: A Key Enabling Tool For Genomics?”. The following represents some of the progresses
made recently.

Automation of 2
-
D Gel Electrophores
is


A patent on an automated system for two
-
dimensional electrophoresis has been issued recently
[4]. In this patent, an integrated, fully automated, high
-
throughput system for two
-
dimensional
electrophoresis comprised of gel
-
making machines, gel processi
ng machines, gel compositions and
geometries, gel handling systems, sample preparation systems, software and methods. The system is
capable of continuous operation at high
-
throughput to allow construction of large quantitative data set
(Fig 2).


However,
this automated 2
-
D electrophoresis is still based on the traditional slab gel
electrophoresis. The fundamental problem associated with the traditional 2
-
D electrophoresis system is
still here. It only reduces human intervention by automating the various
steps. It also involves the gel
making steps in the system, which adds another variable to the system.

Parallel Capillary Electrophoresis Systems

Several commercial companies have produced DNA sequencers based on capillary
electrophoresis (CE) syst
em. PE Biosystems (Foster City, California, USA, 800
-
345
-
5224,
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-
D gel electrophoresis



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www.pebio.com
) has a 96 capillary based ABI Prism 3700 DNA sequencer, which is the centerpiece of the
most genomic research effort. Amersham Pharmacia Biot
ech (Sunnyvale, California, USA, 800
-
526
-
3593,
www.megabace.com
) also has a 96
-
capillary based MegaBACE system. The new player in the 96
-
capillary market is the SCE9600 from SpectruMedix Corp. (State College, Pennsyl
vania, USA, 814
-
867
-
8600,
www.spectrumedix.com
). These 96
-
capillary based CE systems offer faster run times, longer
fragment reads, increased automation, and higher sample throughput [5]. CE expands the throughp
ut by
decreasing the read times and decreasing the maintenance necessary to run the sequencer by eliminating
plate washing, gel casting, and sample loading
-
time consuming tasks common to slab gel systems. In
addition to a 8
-
capillary Paragon CE system for

clinic diagnosis, Beckman Coulter Inc. (Fullerton,
California, USA, 800
-
483
-
5671,
www.beckmancoulter.com
) recently introduces a CEQ 2000 system for
DNA analysis.

These CE systems also eliminates one of the i
ntrinsic problems in slab gels
-
since there is no
physical barrier between lanes, there is always a risk that biomolecules can migrate into neighboring lanes
on a gel. Moving into a capillary system has the practitioners to do much better data tracking sin
ce they
can run multiple samples and not worry about them smearing into each other.


Other than the Beckman Coulter Paragon system, all of those parallel capillary instruments are
capillary gel electrophoresis (CGE) based instrument and are marketed towar
ds the DNA analyses.
However, with minor modifications, all of these instruments can be used for protein analysis [6].
Actually, one of the authors, J. Bao, envisioned the potentials of these CE based parallel separation
systems and made suggestions to S
pectruMedix Corp. to modify their instrument for protein analysis.
With close collaboration, they have successfully modified the SCE9600 system to perform various
capillary zone electrophoresis (CZE) for protein analysis. With the modification, it is pos
sible to analyze
proteins both in CZE and CGE modes. This modification has generated strong interests in pharmaceutical
companies for various analyses including protein separation. Currently, the author is using this modified
system to studying protein i
nteractions and enzyme kinetics [6].


All of these commercial parallel CE instruments offer high degree of automation. The only
draw pack of the current 96
-
capillary systems is that they are still one
-
dimensional system, it has limited
use for proteome

studies due to resolution limit in single capillaries.

Biochips

A new approach to automation and high throughput protein studies involves the use of protein
biochips for analysis of protein expression levels or protein
-
protein interaction [7]. Since t
he chips offer a
platform of two
-
dimensional structure for protein analysis, it is convenient to interface multiple
dimensions of separations with different mechanisms, such as CZE, CGE, or/and IEF. This offers a
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D gel electrophoresis



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capability of performing versatile separat
ions needed for the separation of complex protein samples on a
single surface. It also allows a special selection of proteins for analysis. This capability of “selecting
out” specific proteins allows researchers bypass the problem of identifying low
-
abun
dance proteins and
purification steps needed for MS. Since the chips can identify proteins both by mass (size) and by
binding affinity (CZE or CGE), it can generate information about the activity of proteins of interest. The
most important advantage of
the chips or capillary systems, when compared with the slab gel based
electrophoresis systems, is the extremely small sample volume requirement. This is critical because the
amount of sample available for analysis is often limited and usually multiple ana
lyses are needed for each
sample. By using only tiny amount of sample for the separation analysis, more samples will be remained
for other analyses.

The Biochip technology is relatively new. Despite its many appearing characteristics, such as t
he
capability of less than one
-
minute separation time, there are still several issues associated with this
technology. One major issue is the interface of the microchip platform with the macro platform, such as
the 96 or 384 well based microtiter plates.

How to inject the samples into the channels on the chip
remains a big issue. The detection needs to be more automated. It makes little sense to use a 5
-
cm
separation channel capillary on the chip to perform a separation when we have to use a 50
-
cm capil
lary to
bring the sample into a MS instrument for detection. Even after we have solved the sampling and
detection problems, we still need to work out issues such as protein adsorption, reproducibility,
application to complex samples, and identification of

unknown proteins. However, miniturization and
automation of protein chips may be developed into rapid, automated, and high
-
throughput systems for
protein separation and analysis.

New Integrated 2
-
D Capillary Electrophoresis System

One of the authors, Y.

D. Liu, has developed a new integrated 2
-
D capillary electrophoresis
system for protein analysis [8]. This new system will revolutionize the current practice of the 2
-
D
electrophoresis by combining the two steps and two devices into one single process.

The integrated 2
-
D
CE system can be fully automated to become a turnkey system to allow novice and casual users to
perform the separation and quantitation of proteins on
-
line simultaneously with high accuracy and
precision. This instrument will allow the

seamless transition of proteins separated by isoelectric focusing
(IEF) from the first dimension to the second dimension. The separation in the second dimension can be
achieved either based on molecular weight based (CGE) or based on other interactions,
such as affinity.
This system will also enable the quantitation of the proteins on
-
line without the time consuming and
tedious staining process. It is anticipated that this new system will offer the following advantages:

1)

It allows the detection of prot
eins directly without any staining steps;

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-
D gel electrophoresis



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2)

It enables quantitative determination of proteins with much less background interference;

3)

It can be fully automated;

4)

It is easy for interfacing with other serial techniques, such as LC and MS on
-
line;

5)

It elimi
nates the horizontal diffusion of protein bands.

6)

It maintains the seamless interface between the first and the second dimensions.

7)

It will allow the analysis of proteins in much smaller amount.

8)

The results generated by this new technology may be compared di
rectly with the protein
databases currently available. Further information that gathered through this technology might be
used to revise the current database with more quantitative information.

Currently, the inventor is seeking proper funding for the com
mercialization of this system.

Conclusions



The current practice of using 2
-
D slab gel electrophoresis followed by cutting gels and analyzing
the sample solution by MS needs to be improved with automation to give better and more quantitative
results. Obv
iously, other techniques such as MS will play very important roles in the study of proteomes
[9]. However, protein separation will always be the key to the success of proteomics. HPLC has a long
history for protein separation and will continue to play a
role in the separation of complex protein samples
[10]. The most important technique in protein separation will continue to be electrophoresis. The four
aforementioned new electrophoresis techniques represents the current trend in the development of new
technologies and will play some important roles in future studies of genome and proteome.

Future


As the interest in proteomics continues to grow, it is expected that demand for more efficient and
user
-
friendlier instruments will increase accordingly. It
is expected that there will be a market of 43
billion dollars for proteomics and genomics by 2003 [11]. Many pharmaceutical and biotech companies
are rushing into proteomics as combined genomic and proteomic information can help create a
comprehensive and

dynamic picture of disease mechanisms. For exampme, a drug discovery alliance
concentration on proteomics has been formed between Amersham Pharmacia Biotech and Zeneca
Pharmaceuticals [12]. Rhone
-
Poulenc Rorer (RPR) has signed an agreement with Proteome

Sciences,
UK, and the Heart Sciences Research Center, Harefield Hospital, London, relating to cardiovascular
disease [13]. These kinds of alliance will further help pharmaceutical companies to develop better
instrumentation to meet the need of pharmaceut
ical companies. Actually, investment by pharmaceutical
companies in genomic alliances in 1996 was estimated at $1 billion, up from $720 million in 1995, and
from $320 million in 1994. The well
-
established area is gel electrophoresis sequencing technology.

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-
D gel electrophoresis



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Annual sales of instruments and reagents are estimated at $500 million to $1 billion [14]. For example,
Amersham Pharmacia Biotech is currently developing fluorescent differential gel electrophoresis, which
will be used to detect and identify proteins a
ssociated with disease. In the PRP deal, RPR will fund a
two
-
year research program on the application of proteomics to identify differential regulated proteins in
failing human myocardium. Proteome Sciences uses 2
-
D gel electrophoresis to search for nove
l protein
markers in body fluids and tissues, the presence of which can be used in the development of diagnostics
and treatments. RPR will own the rights to discoveries relating to the treatment of heart disease or
failure, while Proteome Sciences will r
etain rights to diagnostic discoveries. All of these activities
indicate that there is a bright future for the development of new technologies for proteiomic and genomic
studies.

References

1.

M. R. Wilkins, K. L. Williams, R. D. Appel, D. F. Hochstrasser, E
d.
Proteome Research: New
Frontiers in Functional Genomics: Principles and Practice
, Springer
-
Verlag Berlin Heidelberg,
1997.

2.

P. H. O’Farrell,
High resolution two
-
dimensional electrophoresis of proteins
, J. Biol. Chem., 250
(1975) 4007
-
4021.

3.

B. R. Herbe
rt, J. C. Sanchez, and L. Bini,
Two
-
dimensional electrophoresis: the state of the art and
future directions
, in M. R. Wilkins, K. L. Williams, R. D. Appel, D. F. Hochstrasser, Eds.
Proteome
Research: New Frontiers in Functional Genomics: Principles and Pr
actice
, Springer
-
Verlag Berlin
Heidelberg, 1997, pp. 13
-
33
.

4.

N. L. Anderson, N. G. Anderson, and J. Goodman,
Automated system for two
-
dimensional
electrophoresis
, US Patent 5,993,627, Nov. 30, 1999.

5.

J. Boguslavsky,
DNA sequencers reach production scale
, R&D

Magazine, Drug Discovery and
Development, Jan./Feb. 2000, p 36.

6.

J. J. Bao, K. R. Wehmeyer, J. Tu, and C. S. Liu,
High throughput electrophoretic separation system
and its application in pharmaceutical research
, LC
-
GC, in preparation.

7.

J. Boguslavsky,
Prote
omics to play critical role
, R&D Magazine, Drug Discovery and Development,
July, 1999, p 32.

8.

Y. D. Liu,
Method and apparatus for interfacing multi
-
dimensional electrophoresis into an automatic
system for on
-
line analysis
, US Provisional Patent, 1999.

9.

Yates
, John R., III., Mass spectrometry. From genomics to proteomics, Trends Genet. (2000), 16 (1),
5
-
8.

10.

HPLC separation of proteins

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-
D gel electrophoresis



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11.

Proteomics market

12.

European Chemical News, Vol. 70, Issue 1842, Page 25.

13.

Chemistry in Britian, Vol 35, Issue 2, Page 9, Feb, 19
99.

14.

Chemical Market Reporter, Vol. 253, Issue 10 (supplement), Page FR 9., March 9, 1998





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-
D gel electrophoresis



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Figure 1. The elements of proteome technologies


Pre
-
fractionation

& Sample Preparation



2
-
D Electr
ophoresis

Post Separation Analysis

-

Stain/Imaging Analysis

-

Peptide Digestion

-

Mass Spectrometry

Bioinformatics

Database

Drug

Discovery

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-
D gel electrophoresis



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Fig 2. A schematic diagram of the entire automated 2
-
D e
lectrophoresis process.