Proteomics 2002, 2, 3–10
3
Review
Thierry Rabilloud
CEA-Laboratoire de
Bioénergétique Cellulaire
et Pathologique,
Grenoble, France
Two-dimensional gel electrophoresis in proteomics:
Old, old fashioned, but it still climbs up the mountains
Proteomics has traditionnally used the separating power of two-dimensional electrophoresis for the quantitative analysis of protein amounts in complex extracts. However,
the limitations of this approach in terms of throughput and analyzable protein range
have elicited the development of other proteomics approaches, based either on peptide separations instead of protein separations, or based on direct protein recognition
and selection on dedicated arrays (protein chips). These recent methods seem very
promising, and probably look more promising than they will ultimately be, just because
their weaknesses are not fully characterized yet. The purpose of this paper is thus to
highlight the strengths and weaknesses of all the proteomics approaches proposed to
date and to try to deduce the respective niches in proteomics that these approaches
will have in the future.
Keywords: Two-dimensional gel electrophoresis / Mass spectrometry / Arrays / Review
PRO 0120
Contents
1
1 The classical approach:
2-D electrophoresis coupled with mass
spectrometry
The classical approach: 2-D
electrophoresis coupled . . . . . . . . . . . . . . . . . .
3
2
Intrinse limitations of 2-D electrophoresis . . . .
4
3
Approches without 2-D electrophoresis.
What are they, what do they deliever? . . . . . . .
4
3.1
SDS-PAGE coupled with MS/MS . . . . . . . . . . .
4
3.2
Electrophoresis-free, MS-based
approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
3.3
Array-based approaches . . . . . . . . . . . . . . . . . .
6
4
The beauty of 2-D electrophoresis:
Analysis of modified poteins . . . . . . . . . . . . . . .
7
4.1
Analysis of modified proteins
by MS-based methods . . . . . . . . . . . . . . . . . . .
7
Analysis of modified proteins
by array-based methods . . . . . . . . . . . . . . . . . .
7
4.2
4.3
Analysis of modified proteins by 2-D gel
electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Correspondence: Dr. T. Rabilloud, Laboratoire de Bioénergétique Cellulaire et Pathologique, DBMS/BECP, CEA-Grenoble,
17 rue des martyrs, F-38054 Grenoble Cedex 9 France
E-mail: [email protected]
Fax: +33-438-785-187
Abbreviations: ICAT, isotope-coded affinity tags; MUDPIT,
multi-dimensional protein identification technology
ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002
When taking proteomics as the large-scale analysis of
proteins in a cellular-wide frame, 2-D electrophoresis has
dominated the scene for the last 20 years. While 2-DE
was introduced in the mid-70’s [1–3], its real expansion
as a useful technique had to wait for the development of
microanalytical techniques able to identify proteins at the
amounts available from 2-D gels. These microanalytical
techniques were first Edman sequencing [4–6]. More
recently, mass spectrometry has greatly increased the
power of this microcharacterization step, as it is much
more productive and sensitive MS than Edman sequencing [7–9]. In addition, MS also has the power to characterize virtually any type of post-translational modifications
[10]. This combination between mass spectrometry and
2-D electrophoresis has made the turn which resulted in
the word proteomics in the mid-90’s. Since then, multiple
pieces of work using this approach in order to tackle various biological questions have appeared in the literature
([11–15], to quote just a few).
However, it is rapidly becoming evident that this approach
is far from perfect. One of the first weak points comes
from the difficulty in the automation of 2-D electrophoresis. While the post electrophoretic steps are now more
and more robotized, 2-D electrophoresis itself has not
really been automated, and it is highly likely that it will not
be in the near future, although numerous technical
improvements (e.g. the use of immobilized pH gradients)
1615-9853/02/0101–3 $17.50+.50/0
4
T. Rabilloud
have gradually brought the technique from art to craft, but
obviously not to industry yet. In addition to this throughput problem, which is fortunately not critical in many
cases, there are several weak points that really reduce
the utility of 2-D electrophoresis in a cell-wide study, as
shown for example in [16]. These limitations are mainly
due to 2-D electrophoresis itself and/or to the coupling
with mass spectrometry, and are now detailed below.
2 Intrinsic limitations of 2-D
electrophoresis
Although at first glance the resolution of 2-D electrophoresis seems impressive, it is still not sufficient compared to
the enormous diversity of cellular proteins, and comigrating proteins in the same spot are not uncommon [16, 17].
However, the use of zoom or narrow pH range gels will
increase resolution and greatly decrease the probability
of this phenomenon [18].
Much more worrying, however, are other limitations of 2-D
electrophoresis. These limitations are linked to the enormous chemical diversity of proteins and to their very
divergent expression in cells or tissues. The latter problem, which has been reviewed previously [19], can be
very simply exemplified. In standard human cells, the
most abundant protein is often actin, which is present at
ca. 108 molecules per cell. On the other hand, some cellular receptors or transcription factors are probably present at 100–1000 molecules per cell. This makes a 1–105
or 106 dynamic range, which is clearly out of the range of
2-D electrophoresis (max. 104). The situation can be even
worse in some tissues such as serum, where albumin is
present at 40 mg/mL and cytokines at pg/mL levels (109
dynamic range). It must be recalled, however, that this
problem will plague all general approaches of protein
populations. This means that enrichment or prefractionation strategies will be needed to reach the less abundant
proteins, especially in the most complex cells or tissues.
The problems coming from the chemical heterogeneity of
the proteins can be exemplified at two levels. The first one
is the range of pIs and Mrs of proteins, which exceeds
what can be routinely analyzed on 2-D gels, even in very
simple organisms [20]. It can be anticipated, however,
that recent progress in available pH gradients can overcome at least part of these problems [20, 21], and greatly
improve the coverage of the total proteome of a cell [22].
Much worse, however, are the problems linked with protein extraction and solubility during 2-D electrophoresis.
These problems are especially prominent for classes of
poorly water-soluble proteins, such as membrane proteins and nuclear proteins. Here again, recent advances
have improved the situation, but it seems quite obvious
Proteomics 2002, 2, 3–10
that this will remain a major limitation of 2-D based
approaches. In addition, it must be kept in mind that these
problems are linked with the isoelectric focusing step of
2-D electrophoresis and are therefore much less severe
in IEF-free approaches.
3 Approaches without 2-D electrophoresis.
What are they, what do they deliver?
All these limitations have driven several research teams to
propose alternative, 2-D free approaches. As mentioned
in Section 2, all these approaches have in common the
elimination of the IEF step, and either with or without the
SDS-PAGE dimension.
3.1 SDS-PAGE coupled with MS/MS
This approach is the most obvious simplification of the
classical proteomics approach. It can be used under two
main frames. In the first, the analysis is made on a very
simplified fraction, so that it can be expected that a single
SDS-PAGE band only contains one protein. Provided that
a linear gel stain is used between SDS-PAGE and the
mass spectrometry analysis, quantitative data can be
derived. However, this obviously strongly limits the usefulness of this approach to simple biological or biochemical
objects. This approach has been successfully used on the
spliceosome [23] and on a highly enriched chloroplast
envelope preparation [24].
In another frame, much more complex samples are analyzed by SDS-PAGE MS (generally MS/MS). In this case,
each band contains several proteins, so that quantitative
data cannot be derived. The result is thus a list of the proteins present in the starting sample. This means in turn
that quantitative variations among this list occurring
under various biological conditions must be detected by
other means. However, this approach can be very successful when the list of proteins present in the sample is
the goal [25–27].
3.2 Electrophoresis-free, MS-based
approaches
In the SDS-PAGE MS/MS approach just described in
Section 3.1, the SDS-PAGE separation has no other role
than merely deconvoluting the protein mixture, so that the
MS/MS spectra are not overcrowded. However, it must
be kept in mind that although good, the process starting
from a piece of gel and ending in a peptide extract is far
from perfect. The yields are low (commonly 1% or below),
some peptides, especially the large ones (which are
unfortunately those bearing more information) are lost at
Proteomics 2002, 2, 3–10
2-D in proteomics
5
schematically in Fig. 1. In this approach, the total protein
mixture is digested, sometimes by several enzymes, and
the total digest is loaded on a multidimensional peptide
chromatographic separation, interfaced in line with an
MS/MS spectrometer. Of course, the complexity-deconvolution mainly takes place in the peptide chromatography
step, but the capacity of MS/MS to handle several peptides at a time is also used.
An impressive achievement of this approach is its application to the total yeast proteome [29]. The number of
proteins identified by this method is around 1500, while
2-D electrophoresis-based methods have only identified
a few hundreds (ca. 400 spots, corresponding to ca. 300
gene products). In addition, many protein classes are
represented in the MUDPIT proteome, which is not the
case for the electrophoresis-derived proteome. It must
also be mentioned that this approach is amenable to
automation. However, it must be recalled that running a
MUDPIT experiment on a complex proteome requires
smooth operation of a nanospray for extended time periods (at least 20 h) without clogging, which is certainly not
as straightforward as suggested in the paper.
Figure 1. Schematic diagram of the MUDPIT approach
(adapted from [29]). The protein mixture is divided in aliquots, which are each digested by a protease. After protease inactivation, the aliquots are pooled and acidified.
This procedure is intended to maximize the probability
for every protein to be represented by at least one peptide
in the subsequent analysis. The peptide mixture is then
loaded on a strong cation exchanger (SCX) column, followed by a reverse phase column (RP) coupled by a
nanoelectrospray device to a MS/MS spectrometer. The
SCX column is eluted by a KCl step, with the mass spectrometer off. This transfers a population of peptides in the
RP column, where they bind. After an acid rinse to fully
remove the KCl, the RP column is then developed by an
ethanol gradient, with the mass spectrometer on. This
allows identification of the eluting peptides by uninterpreted MS/MS search. When the ethanol gradient is fully
developed, a step of more concentrated KCl is performed
on the SCX column, etc.
the extraction stage etc. This has prompted several
research groups to introduce gel electrophoresis-free
approaches. In this case, the complexity problem is no
longer tackled at the protein level, but at the post-digestion peptide level. A typical example is the multi-dimensional protein identification technology (MUDPIT) approach, introduced in Yates’ laboratory [28] and shown
Moreover, it must be emphasized that in contrast to the
gel-derived proteome, where quantitative data are available both intra-gel (i.e. giving relative abundance data
within a cell) and inter-gel (giving quantitative variations
between different conditions), the MUDPIT-derived proteome is a raw list of proteins present in the sample, without any accurate quantitative aspect. This means in turn
that the only variations which can be identified by this
method are Boolean (present-absent) variations. This
has almost never been seen in classical proteomics, but
can be a bias due to the fact that classical proteomics
does not deal with minor proteins.
Part of the quantitative aspects, i.e. inter-sample comparison, is taken into account in another MS-based approach, isotope coded affinity tags (ICAT) [30], which is
shown schematically in Fig. 2. In this approach, a selection of cysteine-containing peptides is carried out, which
greatly simplifies the subsequent peptide analytical work,
which can be carried out in a single chromatographic step
coupled with MS/MS. The seminal ICAT paper has shown
a comparative, quantitative study [30], but the quantitative reproducibility issue, which conditions the statistical
validity of the data, has not been investigated.
As for the MUDPIT approach, the ICAT approach is theoretically insensitive to the nature of the protein. However,
successful analysis of membrane proteins will require that
cysteines are present in peptides that will not be too
hydrophobic. In addition, it must be recalled that one protein out of seven does not contain any cysteine [17].
6
T. Rabilloud
Proteomics 2002, 2, 3–10
3.3 Array-based approaches
In these approaches, specific protein recognition molecules are deposited on a surface in a regular manner.
These molecules bind to their protein ligands in the complex extract to be analyzed (for a review see [31]. The
binding has then to be detected. Generally, in contrast to
what is carried out with DNA arrays, the ligands are not
labelled and detection occurs through the binding of
another protein binder, which is labelled. Alternatively,
more direct detection means, such as MS scanning of
the array [32] or surface plasmon resonance at the surface of the array [33, 34] can be used.
Figure 2. Schematic diagram of the ICATapproach (adapted
from [30]). The ICAT probe is composed of a biotin tag, a linker and a iodoacetamide handle. The linker can be made in a
light version (8 hydrogen atoms) or in a heavy version (8 deuterium atoms), leading to two probes with indistinguishable
chemical behavior but with a mass difference of 8 Da. When
the comparative analysis of two extracts is made, one
extract is labelled (on the cysteines) with the light probe
(black dots), while the other is labelled with the heavy probe
(gray dots). The two extracts are then pooled and digested
with trypsin. The digest is then loaded onto a column of
monomeric avidin to retain the labelled, cysteine-containing
peptides. They are eluted from the avidin by formic acid. The
eluate, which consists of peptide pairs (heavy and light) is
then loaded onto a separation medium (RP chromatography
or capillary electrophoresis) coupled by a nanospray device
to a MS/MS spectrometer. At the first MS stage, each peptide appears as a mass pair separated by 8 Da (provided
there is a single cysteine per peptide). As everything after
labelling has been made on pooled extracts, the yields are
the same and the mass signal intensity ratio gives the ratio
between the two peptides, i.e. between the two conditions.
Fragmentation and MS/MS peptide identification leads to
the name of the corresponding protein. Note however that
the cysteine content differs widely from one protein to
another, so that some proteins will be very nicely assigned
by several peptides, while the analysis of others will rely on a
single peptide. Note also that not every labelled peptide
appears in the final analysis, as peptide losses are not rare in
these wide-range, low-scale peptide analysis schemes.
Note also that some proteins are cysteine-free and remain
unanalyzed by this technique.
The most obvious choice in this scheme is to use antibodies or derivatives thereof (e.g. single chain variable
fragments) as protein recognition molecules, very similarly to what is carried out in a regular sandwich assay.
In this case, the challenge is first to have enough specific antibodies to be able to perform this type of analysis on a large-scale basis. At such a scale, classical
antibody production in animals may no longer be a
good choice, even on a monoclonal basis. Thus, alternative methods, such as phage display, are currently
favored for antibody production [35, 36]. Even if such
a collection of antibodies (several thousands) can be
generated, other problems immediately arise. This type
of assay can be viewed as several thousand of immunoassays performed on a microscale in parallel. The problem of antibody specificity will probably be a major
issue, as it is already the case in the development of
specific immunoassays. These issues have prevented
such a protein array to be implemented yet. However, in
the perspective of this paper, which is the comparison
with 2-D gels and MS-based methods, we can imagine
that these arrays methods work, and try to anticipate
what they can deliver. Despite what is frequently encountered in MS-based methods, the array methods are
easily quantitative and precise, as standard immunoassays already are, even if there is a loss in performance
due to the large-scale and suboptimal process. If we
start from what is already achieved by current immunoassays, the methods should also be more sensitive
than 2-D gels, and should not display any bias against
extreme pI and Mrs. However, the scope of able to be
analyzed proteins should also be limited, especially in
the case of membrane proteins. If we take the example
of immunoassays, it is obvious that very harsh but efficient extraction solutions (e.g. SDS) cannot be used, as
they will damage the antibody. It can be said that milder
extraction of membrane proteins is common practice.
The problem then turns into the quantitative extraction
of membrane proteins by mild solutions, which is, by
far, not guaranteed [37].
Proteomics 2002, 2, 3–10
2-D in proteomics
7
4 The beauty of 2-D electrophoresis:
analysis of modified proteins
4.1 Analysis of modified proteins by MS-based
methods
As has been shown before, 2-D gel electrophoresis
stands out by its remarkable quantitative analysis capabilities. In addition, 2-D gel work is highly suited when
post-translational modifications have to be studied. In
most cases, the proteins are modified only at a few sites.
Consequently, most digestion-derived peptides are
exactly the same in the unmodified and modified forms
of the proteins. This ensures correct identification of the
protein in the databases without requiring too much peptide sequencing effort. However, this also means in turns
that the sequence coverage of the protein, i.e. the portion
of the protein covered by the peptides analyzed in MS, is
absolutely critical when working with post-translational
modifications. While it is true that excellent sequence
coverage can be achieved [38], it must be emphasized
that this is not the average situation in the MUDPIT
approach. The situation is probably even worse in the
ICAT approach, due to the low cysteine content of most
proteins.
To circumvent these problems, some very nice approaches have been recently introduced for the detection of phosphorylated proteins [39, 40]. One of these
approaches is schematized in Fig. 3. It can be easily seen
that this approach requires fine chemistry of microquantities of peptides, which is certainly not straightforward. In
addition, these approaches also suffer from the quantitative drawback. This means that they will just deliver a list of
phosphorylated proteins in the conditions under investigation. However, if a quantitative change in phosphorylation is the important phenomenon (e.g. in [41]), these methods will not be adequate. In addition, these chemical
methods are well suited for phosphate groups, where
selective modifications can be easily carried out. For other
modifications, such as acylations, a chemical scheme will
be much more complicated to design, if ever possible. In
the case of peptide cleavage, the identification of the
cleavage will require that a peptide analyzed in the
MUDPIT or ICAT scheme is either missing or modified by
the cleavage. Otherwise, both the uncleaved and cleaved
form of the protein will be recognized as a single entity.
4.2 Analysis of modified proteins
by array-based methods
In array-based techniques, the recognition of modified
forms requires that one of the protein recognition molecules either does or does not recognize the modified
Figure 3. Schematic diagram of the phosphopeptide
approach by biotin labelling (adapted from [39]). The starting point is the mixture of peptides arising from the trypsin
digestion of a protein extract (which can be a very complex
extract), in which the cysteines have been alkylated. For the
sake of simplicity, only one phosphopeptide and one
unphosphorylated peptide have been represented. The
amino groups, which can interfere with the reaction process, are protected (first step) with a BOC (tert-butyl oxycarbonyl) group, which is removed at the final deprotection
step. The basic idea is to chemically derivatize the acidic
groups, and to use the slight differences in chemical reactivity to achieve selective labelling of the phosphate groups
with a biotin probe. This enables selection of phosphopeptides only on an avidin column. Deblocking ensures
proper elution of the native phosphopeptide, with all grafted
groups cleaved away. MS/MS analysis enables identification of the peptide, and thus of the starting protein, and
in some instances, of the phosphorylation site. Note the
complexity of the reaction scheme (8 steps) to be carried
out on small quantities of a complex mixture of peptides.
Note, however, that the unphosphorylated peptides will act
as carrier molecules and help in minimizing losses.
form of the protein. A classical case when using antibodies is the use of antiphospho-amino acid antibodies.
The performance of these antibodies is described as
correct for antiphosphotyrosine, but still seems rather
poor for antiphosphoserine and antiphosphothreonine
antibodies [42]. Even if major improvements are regularly
claimed for this method, it is highly likely that these antibodies will miss phosphorylations. A good example is
8
T. Rabilloud
human trypsin, which is phosphorylated on a tyrosine
[43]. This has remained completely unnoticed up to the
protein crystallographic stage, and subsequent analysis
by blotting using antiphosphotyrosine antibodies has
not proved able to demonstrate this phosphorylation
(T. Rabilloud, unpublished data). Here again, it must be
kept in mind that phosphorylation is one of the bestsuited post-translational modifications. For example, detection of peptide cleavage requires us to predict the
cleaved peptide and to derive an antibody for each predicted cleaved peptide. Detection of smaller chemical
moieties, such as acylated amino acids, will also be very
difficult, as the structural modification made to the side
chain is very minor and may escape the resolving power
of antibodies.
4.3 Analysis of modified proteins by 2-D gel
electrophoresis
In this case too, the basis of the recognition of modified
proteins in 2-D gel electrophoresis is very simple. One of
the separation parameters (pI and Mw) must be altered in
the modified form. Although there is always a mass alteration, it is generally too small to be detected in the SDS
dimension. The only exceptions to this rule is the artefactual mass increase sometimes encountered in phosphorylation and the mass decrease seen when a rather large
peptide is cleaved. Thus, the parameter leading to the
detection of modifications is most often the pI. This
means in turn that modifications that do not alter the pI
(e.g. cysteine alkylation or acylations) will remain unnoticed. However, many modifications alter the pI, such
as phosphorylation, of course, but also lysine acylation,
Asn-Gln deamidation etc. When such modifications are
detected by 2-D, two important features must be underlined. The first is that 2-D electrophoresis will provide a
quantitative estimate of the extent of modification, but
will treat all equivalent modifications at the same level,
independently of their site on the protein. A very good
example is represented by stathmin, a small protein
that can be phosphorylated on several sites [44]. 2-D gel
analysis will provide a quantitative example of the non-,
mono-, di-, tri-, and tetra-phosphorylated forms. However, it will not be possible by this analysis to distinguish
forms that are monophosphorylated, but on different
amino acids in response to different kinases.
The second very important feature, is the fact that the
sequence coverage in 2-D electrophoresis is 100%. On
the one hand, this can lead to grouping of different protein
forms, as just shown above. But on the other hand, it can
also point out to very discrete modifications, wherever the
modification takes place on the protein. For example in
the case of peptide cleavage, the presence in the cleaved
Proteomics 2002, 2, 3–10
peptide of a single charged residue will be sufficient to
detect cleavage, whether the cleaved peptide is long or
not and located at the N- or C-terminal part of the molecule. In such cases of subtle, but pI-detectable modifications, the classical 2-D gel electrophoresis-mass spectrometry couple is almost ideal. The 2-D gel will point to the
modification and allow its quantitative analysis, while the
MS analysis will allow the precise determination of the
modification. In addition, if this modification is important
in the biological phenomenon under study, the fact that it
has been pointed out by 2-D electrophoresis will drive the
scientist to make the necessary effort to discover its
details. This means that dedicated, unclassical, MS
approaches can be implemented in such cases to get
into the intimate details of the protein modification.
Some examples of detailed posttranslational modifications have been described [10].
5 Conclusions
It is quite obvious that there is now a blossoming of new
analytical techniques for proteomics analysis, and that
the dominant approach based on 2-D gel electrophoresis
is now seriously challenged by other approaches
described in this paper. This may alleviate many technological limitations encountered by 2-D electrophoresis
and allow us to focus an the real biological questions
which we want to solve by proteomics. As a matter of
fact, 2-D or not 2-D, that is NOT the question. The real
question is rather when and why go for 2-D gels or avoid
them. Very short guidelines along this trend will be provided now.
If the goal of the proteomic experiment is to provide a list
of the proteins present in the sample of interest and/or to
select markers which are specifically present under some
biological circumstances, there is little doubt that MS/MS
based approaches, such as the one coupled to SDSPAGE or the MUDPIT approach, will prove far superior to
any 2-D gel-based approach. For these purposes, the
ICATapproach seems somewhat inferior, due to the lesser
number of peptides analyzed and to the number of
cysteine-devoid proteins. At this point in time, the real
performance of arrays remains difficult to predict. However, if the goal of the proteomic experiment is to look for
quantitative changes in the biological process of interest,
and/or to look quantitatively at protein modifications that
can be unclassical, then 2-D gels will remain unrivalled for
some time, as they have the potential to look at unexpected phenomena. It should be emphasized that due to
the relatively low number of genes encountered in upper
eukaryots, an important part of the diversity in the cells
will certainly come from quantitative changes in the
Proteomics 2002, 2, 3–10
2-D in proteomics
9
Figure 4. Comparative 2-D gel analysis of a total cell extract and of a mitochondrial extract. Equivalent amounts (100 mg) of proteins coming from either total Jurkat cells or from mitochondria
extracted from Jurkat cells were loaded on two dimensional gels. First dimension: IPG pH 4–8 (linear);
second dimension: SDS-PAGE (10%T); detection by silver staining. Equivalent proteins are labelled
on the two gels. Note the massive enrichment obtained by the mitochondria preparation (e.g. AOP1,
antioxidant protein-1), and the depletion in major cytoplasmic proteins (e.g. tubulins). This means in
turn that the minor spots visible in the mitochondrial gel are either undetected or masked by a more
abundant cellular protein in the total extract gel.
expressed gene repertoire, and not just from selective
gene expression. This situation points to our need for
methods allowing us to obtain precise quantitative data.
For the moment, only 2-D gels have shown their ability
in this area. The real performance of array-based
approaches and of ICAT remain to be assessed. In addition, the scope of phenomena that can be studied by
2-D gels is rather large. The only constraints that 2-D
gels have are that the phenomena of interest must modify
either the pI, or the Mw, or the abundance of the protein,
and that the protein(s) of interest can be detected on the
2-D gels. This is certainly the major challenge this technology has to face, both at the protein abundance level
and at the protein nature level. As to protein abundance,
the combination of narrow pH gradients and sample prefractionation can lead to an improvement by at least one
order of magnitude over the current situation of analysis
of a total cell extract on a single gel. An example of the
benefits of sample prefractionation is shown in Fig. 4,
which shows the comparative analysis of a total cell
extract and of amitochondrial extract. The improvement
in terms of analysis depth (i.e. minor proteins) and in
terms of pattern simplification (removal of major cellular
proteins) is absolutely obvious. Thus, the major challenge
we have to face with 2-D electrophoresis is the analysis of
extreme proteins (extremely basic or acidic, extremely
small or big, extremely hydrophobic). Looking back at
the progresses that has taken place in the last five years,
there is little doubt that the situation will continue to
improve.
The final message of this paper will be very simple. When
we will have the lists of the proteins present in our samples, as delivered by MS-based approaches, maybe we
will use arrays to get quantitative data (but maybe not).
But, in any case, when we will have to go into the real protein complexity, as exemplified by post-translational
modifications, we will most probably go again to good
old 2-D gels. So when it will be time to go back to the
future, 2-D gels will blossom again.
Received May 31, 2001
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