402
From the analysis of protein complexes to proteome-wide
linkage maps
Pierre Legrain*†, Jean-Luc Jestin# and Vincent Schächter*‡
Recent advances in genomics have led to the accumulation of
an unprecedented amount of data about genes. Proteins, not
genes, however, sustain function. The traditional approach to
protein function analysis has been the design of smart genetic
assays and powerful purification protocols to address very
specific questions concerning cellular mechanisms. Lately, a
number of proteome-wide functional strategies have emerged,
giving rise to a new field in biology, proteomics, that addresses
the biology of a cell as a whole.
Addresses
*Hybrigenics, 180 Avenue Daumesnil, Paris 75012, France
† e-mail: plegrain@hybrigenics.fr
‡ e-mail: schachter@hybrigenics.fr
# Unité de Chimie Organique, Institut Pasteur, 25 rue du Dr Roux, Paris
75724 Cedex 15, France; e-mail: jjestin@pasteur.fr
Current Opinion in Biotechnology 2000, 11:402–407
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
2DE two-dimensional gel electrophoresis
MS mass spectrometry
Introduction
Several technologies to study specific cellular functions or
processes have been around for many years, such as enzy-
matic assays, complex purification or subcellular
localization. In most cases, the studies have focused on a
small set of genes or proteins. The increasing amount of
genomic data, however, has led to the availability of more
new biological objects than could reasonably be studied
using classical genetic or biochemical means. An initial
approach was to screen for new essential genes, assuming
that these genes would be more interesting than others. It
soon became clear, however, that most genes could be
deleted without obvious changes in phenotype and that it
was necessary to study combinations of proteins or subtle
phenotypes to understand cellular functioning. Geneticists
had been studying mutant phenotypes and grouping genes
to build pathways. New technologies were developed,
such as the yeast two-hybrid or phage-display assays that
allow the detection of protein–protein interactions in order
to build protein interaction maps, or synthetic lethality
screens that identify genes whose products are functional-
ly related. Biochemists have purified proteins associated
with an activity in order to characterize the components of
biochemical complexes. Two-dimensional gel elec-
trophoresis (2DE) was coupled to protein mass
spectrometry (MS) analysis to characterize the components
of a complex or even to identify exhaustively most compo-
nents of an expressed proteome, yielding protein
expression maps. More recently, bioinformaticists devel-
oped algorithms to group proteins according to their
sequences assuming that such clustering of proteins would
lead to functional grouping (see below for discussion). It is
expected that the combination of several of these tech-
niques applied at a proteome scale will lead to completely
different approaches for functional analysis, integrating
enzymatic complexes and cascades (e.g. metabolic path-
ways or maps) in a cell architecture and leading to an
integrated view of cell functioning. We present here vari-
ous approaches to link proteins together in order to build
functional networks.
Protein interaction maps
Two-hybrid in yeast
Since its original description in 1989 [1], the yeast two-
hybrid assay has been used extensively to identify
protein–protein interactions. Initially designed as an
assay for the detection of an interaction between two
known proteins, it was rapidly developed as a screening
assay to find partners for a protein of interest [2]. There
are currently many variations around the same principle
(for reviews see [3–5]). In most cases, the goal has been
to find partners for one or several proteins and to build
hypothesis-driven experiments from the two-hybrid data.
Recently, several groups have developed strategies that
allow construction of functional networks based on pro-
tein interaction maps [6–8]. These approaches have
attempted to solve some of the technical drawbacks
inherent to the two-hybrid approach, namely false posi-
tives (proteins that interact non-specifically) and false
negatives (protein–protein interactions that are not
detected in a classical yeast two-hybrid approach), in
order to improve the quality of the data obtained as well
as to provide as complete a description as possible of the
protein–protein interactions occurring within a cell.
Indeed, yeast two-hybrid strategies have been used suc-
cessfully to decipher both stable biochemical complexes
[9,10] and metabolic pathways, such as cell-cycling or
splicing [6,7,11•]. With the recent tremendous increase in
genome sequence data (with still more to come) it
becomes attractive to consider these protein interaction
maps as a first step toward the analysis of protein func-
tion. Indeed, genome-wide approaches have been
reported on T7 phage [12], yeast [7,11•,13•,14•], the
human hepatitis C virus [15•] and Caenorhabditis elegans
[16•]. How these various experimental approaches will
compare to each other is still an open question. As a
whole, however, they will provide the scientific commu-
nity with efficient and accurate tools to approach protein
function on a large-scale and in a systematic way. In addi-
tion, they will help pinpoint many more proteins as new
potential targets for drugs, while protein–protein interac-
tions themselves could become promising targets for drug
design in their own right [17].
 From the analysis of protein complexes to proteome-wide linkage maps Legrain, Jestin and Schächter
Other in vivo two-hybrid assays
During the past few years, several two-hybrid assays have
been developed in organisms other than yeast. In order to
build protein-linkage maps, a sufficient throughput at a
cost-effective level must be achieved. In this respect,
Escherichia coli appears potentially as an even more suitable
host than yeast for protein–protein interactions screens.
Indeed, a transcriptional activation assay in E. coli was pub-
lished, very similar to the yeast assay, showing that a
simple contact between a DNA-bound protein and the
RNA polymerase mediated through two arbitrary polypep-
tides could elicit transcription [18]. Two other assays have
been published based on the reconstitution of an enzyme,
leading to a measurable enzymatic activity [19] or to the
activation of a signal transduction pathway [20].
Although these technologies are not yet applicable to
screening for protein–protein interactions, one of them has
been used for screening variants in leucine-zipper libraries
[21] by reconstituting an active DHFR enzyme made of
two separate inactive polypeptides through a leucine-
zipper structure. Despite the absence of many post-transla-
tional modifications in bacteria, such systems might present
many advantages for high-throughput screening compared
to yeast: the protein complex is built in the
cytoplasm, not on DNA, avoiding transcriptional autoacti-
vation; also, screening assays might be more cost effective
because of generaton time of bacteria as well as easier and
inexpensive molecular biology on E. coli compared to yeast.
Protein–protein interactions can also be screened in mam-
malian cells, allowing a more natural environment for
many metazoan proteins [22]. This assay may or may not
be amenable to large-scale screening.
Phage display technologies
Phage display provides a physical link between a polypep-
tide and its coding gene. The polypeptide is expressed as a
fusion with a phage coat protein on the surface of the phage
particle, which contains the corresponding gene fusion.
The phage-displayed polypeptide can be selected through
interaction with a target using affinity chromatography and
further characterized by amplification and sequencing of
the corresponding gene. Although no protein–protein inter-
action map using phage display has been established so far,
the technology might well have the necessary scaling
potential: cDNA libraries have been successfully expressed
on phage T7 [23], filamentous phage [24] and phage lamb-
da [25], thereby providing a means to identify proteins
interacting in vitro with given targets. An E. coli genomic
library displayed on filamentous phage has also been con-
structed and tested in a model system [26].
Protein expression maps
Proteome-wide protein identification
Protein expression maps aim to identify proteins localized
in specific protein complexes, in organelles or in cells. A
typical approach consists of the separation of the various
403
proteins of an extract by gel electrophoresis followed by
mass spectrometric analysis of protein gel spots, providing
precise identification of polypeptides by unique assign-
ments with their corresponding DNA sequences through
the use of sequence databases [27]. Recent optimizations
of the various steps have provided one of the most power-
ful approaches in proteomics.
First, accurate purification method(s) for proteins, protein
complexes or organelles are required. These mainly
include centrifugation in density gradients [28,29], exclu-
sion chromatography [30], and affinity chromatography
using, for example, peptide tags [31], antibodies (immuno-
precipitation) [32,33•,34] or substrates [35]. A tandem
affinity purification (TAP) involving a combination of two
high-affinity tags linked to the protein of interest permits
a very efficient purification in two steps from a crude
extract [36•]. The technique has been suggested as a gen-
eral method for protein complex purification under mild
conditions after expression in a natural environment [36•].
It remains to be demonstrated that this technique is
applicable to organisms other than yeast.
Second, sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS–PAGE) is typically combined with
isoelectric focusing (IEF) to separate the various pro-
teins according to their isoelectric point on a gel with an
immobilized pH gradient to provide a 2DE technique,
which is thus far the most commonly used multidimen-
sional protein separation method used in proteomics
[32,33•,35,37]. Recently, innovative sample preparations
have made membrane-associated proteins amenable to
2DE analyses [38]. As this technique is becoming stan-
dardized and reproducible in different laboratories,
annotated databases of 2DE images have been created
for various proteomes [37,39].
Third, the separated protein spots on the gel can be
excised and the proteins proteolytically digested in-gel.
Peptides are subsequently eluted from the gel and
analysed by MS. High throughput is achieved by auto-
mated matrix-assisted laser desorption/ionization
(MALDI), providing a list of masses for the peptides.
Matching this list against the list of calculated peptide
masses from an appropriate protein sequence database
characterizes the isolated protein. This method alone
was found to be sufficient in several proteomic
studies [28,33•].
Electrospray ionization (ESI) MS requires a preliminary
peptide purification step and provides both the peptide
mass list (ESI MS) and amino acid sequences of select-
ed peptides on tandem mass spectra (ESI MS/MS): this
allows unambiguous identification of a protein’s
sequence by database searching [29–32,35]. High-
throughput methods have been designed to identify
various post-translational modifications of proteins
by MS [40•].
404 Protein technologies and commercial enzymes
Proteome-wide protein quantification: towards
differential maps
Further proteome-wide characterization, which requires
accurate large-scale quantification, allows the production
of global maps of differentially expressed proteins.
Radioactive labeling of metabolites yields protein extracts
that can be analyzed quantitatively on 2D gels by scintilla-
tion counting [41]. Stable isotope metabolic labeling of
distinct protein pools followed by MS analysis of proteins
purified from 2D gels provides a further method for quan-
titative and differential analysis of protein expression [42].
An analogous strategy consists of direct MS analysis of
purified proteins: cell extracts are treated with alkylating
reagents which are tagged for affinity isolation and labeled
by different stable isotopes for MS characterization of the
distinct cell types or cell states studied [43••]. This strate-
gy does not make use of 2DE technology and thereby
avoids its main limitation, that is, its inability to detect and
quantify rare proteins and the difficulty of comparing gels.
Differential protein expression maps have been applied,
for example, to the elucidatation of signal transduction
pathways [42,44], the characterization of distinct cell types
[45,46], the identification of virulence factors of pathogen-
ic bacteria [47], parasite-encoded membrane-proteins [38],
and proteins specifically associated with human diseases
[48••], and the determination of the effects that environ-
mental changes have on protein expression [43••,49].
Other protein linkage maps
Transcriptome maps
The availability of completely sequenced genomes has
permitted the production of microarrays of all the predict-
ed genes of a particular organism. By performing
hybridization experiments with probes prepared from
RNAs of cells grown under varying conditions, it has been
possible to produce genome-wide quantitative patterns of
gene expression, and by inference maps of co-expressed
proteins. Protein expression does not, however, always par-
allel mRNA expression [41]. In addition, different
techniques used in gene expression profiling are far from
being directly comparable to one another. Nevertheless,
we believe that standardized and systematic studies on
gene expression will provide rich foundations for further
functional studies.
Phenotypic mutant linkage maps
The availability of completely sequenced genomes also
paves the way for systematic gene disruption experiments.
A list of essential genes can be compiled [50•], and more
importantly, systematic analysis of viable disrupted
mutants allows the grouping of genes according to mutant
phenotypes [51•,52••]. Such studies have been performed
not only on small bacterial genomes and yeast, but also on
higher eukaryotes with a collection of tagged mouse
embryonic stem cells [53]. This should ultimately lead to
databases of mutant phenotypes directly related to a given
mutant genotype.
Prediction-based linkage maps
Whereas the accumulated knowledge on protein linkage
maps has been derived so far mainly from direct exploita-
tion of biochemical and genetic experiments, recent
approaches attempt to bypass the collection of specific
experimental data altogether by predicting functional
links between proteins through computational means.
These approaches are founded on various biological
hypotheses (phylogeny, structure, etc.) and tested on dif-
ferent sets of experimental data (sequences, gene
expression, etc.), this data being available in databases of
various kinds, bibliographical included.
Promising attempts to automatically extract protein–pro-
tein interaction information from scientific text, for
example, by applying parsing techniques based on a
restricted vocabulary to Medline abstracts [54•], need to be
refined in accuracy and scaled-up to produce sizeable
maps. Other works endeavor to identify functional links
based on the ordering of related genes on genome
sequences, following the notion that gene proximity is a
result of selective pressure to associate genes that are co-
regulated and thus potential interacting partners [55,56].
The so-called ‘domain-fusion’ method [57•,58] also relies
on sequence data to predict interactions; it is based on the
idea that a configuration where a protein (the ‘composite
protein’) contains two domains orthologous to two full-
length proteins of a given species (the ‘component
proteins’) hints at the existence of an interaction between
the component proteins. Although comparisons with bio-
chemical data obtained independently validate each of
these prediction schemes to a certain degree, all have the-
oretical limitations, and false negatives as well as false
positives abound. Furthermore, the predicted functional
links may correspond to indirect functional associations,
such as involvement in the same pathway or complex, as
well as to direct molecular interactions. Given the present
state of the art, additional information or biological valida-
tion is thus required to reach conclusive evidence on both
the existence and the nature of a given functional link.
One way to reduce this uncertainty is to combine inde-
pendent prediction methods, or better yet, methods based
on different types of experimental data. Links between
proteins showing similar phylogenetic profiles, correlated
mRNA expression patterns, or which participate in the
same pathways have been compounded with domain-
fusion predicted links, resulting in a higher confidence
composite linkage map [59••].
Conclusions
Proteomics aims to determine the nature and the quanti-
ty of proteins present in biological samples, to identify
linkage between proteins and ultimately to understand
the function of these proteins. The approaches
described in this review — ranging from yeast two-
hybrid or phage-display assays for interaction map
construction to the combined use of 2DE and MS for
protein expression map building, to the faster but less
 From the analysis of protein complexes to proteome-wide linkage maps Legrain, Jestin and Schächter
conclusive computational prediction techniques —
evolve from different technological backgrounds, but all
stem from an effort to scale up from ad-hoc, ‘local’ pro-
tein analysis to global, proteome-wide pictures.
Such a change in scale — a prerequisite to the develop-
ment of industrial applications in the fields of drug
discovery and diagnostic tests — impacts both the nature
and the organization of proteomics work.
The production of proteome-wide linkage maps relies not
only on high-throughput experimental technologies, but also
on extensive bioinformatics support. For example, protein
identification from 2DE experiments involves searching
sequence databases, storing annotated gel images, identify-
ing spots using sophisticated image analysis techniques, as
well as computing and tabulating molecular weights from
mass spectra. Efficient storage, manipulation and retrieval of
experimental data, as well as the integration of technology-
specific algorithms in a seamless chain of data transformation
running from raw experimental data to exploitable results,
are thus essential enablers of the production process.
As the larger proteomics projects demand resources sel-
dom available to isolated academic or industrial
laboratories, distribution of work and pooling of resources
within mixed academic/industrial networks will become
increasingly necessary. Such collaborative efforts will
revolve around the sharing of experimental data and post-
analysis results, as yet nonexistent common databases will
integrate data produced by several groups using different
or even totally unrelated techniques.
If these data are to be related meaningfully, the establishment
of experimental standards for quality, of common computer
representations for both experimental protocols and results,
and of a set of corresponding benchmarks, are likely to
become major issues within the proteomics community.
Another essential enabler for proteomics research should be
the development of Internet-based graphical interfaces for
the visualization, exploration and analysis of linkage maps
resulting from each type of technology [60]. As front-ends for
databases following adequate standards of quality and pre-
sentation, these interfaces may provide not only a popular
novel medium for the dissemination of scientific results, but
also a mandatory complement to more traditional publishing.
Finally, meaningful integration and useful presentation of
linkage maps of the same biological nature obtained
through diverse means is only the first step towards the
fulfillment of the promise of proteomics. Beyond ‘single
data-type’ representation, analysis and visualization tools,
one can envision more ambitious frameworks aimed at the
comprehensive study of ‘biological links’ resulting from
several distinct data types. Such compound, generalized
linkage maps should be fertile grounds for the discovery of
more global insights into the molecular mechanisms of life.
405
Acknowledgements
We thank D Strosberg, S Whiteside and J Wojcik for critical review of the
manuscript. We are also grateful to A Danchin for many stimulating discussions.
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