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Integrating forward and reverse proteomics to

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Proteomics 2006, 6, 5467–5480 DOI 10.1002/pmic.200600211 5467

REVIEW

Integrating forward and reverse proteomics to unravel

protein function

Sandrine Palcy1 and Eric Chevet1, 2*

1
Organelle Signaling laboratory, Department of Surgery, McGill University, Montreal, Quebec, Canada
2
Departments of Anatomy and Cell Biology and of Medicine, McGill University, Montreal, Quebec, Canada

To date, proteomics approaches have aimed to either identify novel proteins or change in protein Received: March 28, 2006
expression/modification in various organisms under normal or disease conditions. One major Revised: May 19, 2006
aspect of functional proteomics is to identify protein biological properties in a given context, Accepted: May 21, 2006
however, forward proteomics approaches alone cannot complete this goal. Indeed, with the
increasing successes of such proteomics-based research strategies and the subsequent increas-
ing amounts of proteins identified with unknown molecular functions, approaches allowing for
systematic analyses of protein functions are desired. In this review, we propose to depict the
complementarities of forward and reverse proteomics approaches in the definite understanding
of protein functions. This dual strategy requires a data integration loop which allows for sys-
tematic characterization of protein function(s). The details of the integrative process combining
both in silico and experimental resources and tools are presented. Altogether, we believe that the
integration of forward and reverse proteomics approaches supported by bioinformatics will pro-
vide an efficient path towards systems biology.

Keywords:
Bioinformatics / Functional genomics / High-throughput proteomics / Protein

1 Introduction formation [2]. In addition, the identification of novel proteins

Proteomics has become a major discipline not only in the
field of biology, applying to biochemistry, cell, and molecular
biology but also to pharmacology and toxicology. This dis-
cipline, which mainly relies on technological advances at the
level of protein separation, identification, sensitivity or
throughput, and on bioinformatics is essentially based on
the understanding of changes occurring at the level of the
proteome in cells, tissues, or organisms [1]. These changes
may include not only quantitative parameters such as
expression levels but also qualitative aspects such as locali-
zation, post translational modifications (PTM), or complex
Correspondence: Dr. Sandrine Palcy, Mcgill Surgery, 687 Pines
Av. West, Montreal Quebec H3A1A1, Canada
E-mail: palcy@hotmail.com
also represents an avenue for the understanding of yet
unclear biological mechanisms.
For such accurate characterization, one requires not only
the capacity of generating quantitative data but also to assess
the diversity of the above-described parameters at a pro-
teome-wide scale. The sequencing of an increasing number
of genomes added to the large cohort of proteomics data have
allowed the generation of reliable databases which in turn
facilitate the acquisition of accurate and significant data at
the level of the prime proteome analysis. Quantification
represents also an important parameter in the design of
proteomics experiments to provide data which in turn could
be utilized to model specific biological systems.
These approaches defined as forward proteomics have
been successfully integrated in an increasing number of
studies describing proteome-wide model systems [3, 4]. In
addition, in several studies this information was also inte-

Abbreviations: ICAT, isotope-coded affinity tags; SCX, strong cat- * Additional corresponding author: Dr. Eric Chevet
ion exchange E-mail: eric.chevet@mcgill.ca

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

5468 S. Palcy and E. Chevet
grated to other types of datasets such as transcriptome [4] or
localizome [5, 6] thus providing more comprehensive repre-
sentations of defined models and their variations.
However, although these proteome-wide approaches have
brought a very significant amount of biological data informa-
tive for specific biological systems, the biological functions of
specific individual components of these systems remain diffi-
cult to assess in a comprehensive manner. In a context where
biological systems can be decomplexified as simpler parts, it
becomes of interest to develop integrated experimental
approaches in which these simplified systems are not only
characterized by the identification of their protein content (and
its variation(s)) but also by the functional characterization of
each individual protein identified in relevant experimental
models (from in vitro to in vivo; reverse proteomics). A pre-
conceived experimental design which will take into account
the nature and composition of the initial biological system will
therefore allow major progresses in the understanding of the
specific functions of each individual component.
2 Description of forward and reverse
proteomics
Thus far, proteomics has been mainly considered as an
approach allowing for either protein identification from
complex mixtures or the characterization of changes at the
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 5467–5480
level of their expression/PTM levels. This has been named
forward proteomics and relies mainly on the power of sam-
ple preparation technologies, MS analyses and bioinfor-
matics to achieve satisfactory goals. However, the under-
standing of protein function(s) has necessarily to be carried
out using recombinant-based methodologies. These func-
tional approaches which include recombinant expression in
various systems, i.e., for protein localization and functional
characterization, can be named reverse proteomics when
based on an initial biological system. In this context, the
biological information associated to each component of the
system studied can indicate a specific modularity, which as a
consequence, may determine its functionality as a whole.
Consequently, forward proteomics is used to define (exhaus-
tively) the components of a given biological system and
reverse proteomics to study in an integrated manner the
biological characteristics of this system.
2.1 Forward proteomics
This refers to the classical proteomics approach starting
from a protein sample, and then leading to the characteriza-
tion and identification of its content. The process flow
described in Fig. 1 reports a number of critical steps to con-
sider in forward proteomics approaches. Initially, a critical
Figure 1. Schematic repre-
sentation of the forward prote-
omics workflow.
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Proteomics 2006, 6, 5467–5480
decision step is made at the level of the separation method
which will condition both the characterization methods and
the identification steps (Fig. 1).
2.1.1 Protein isolation/extraction, separation, and
preparation for MS analyses
Regardless of the strategy chosen (global or hypothesis-driv-
en analyses), the nature of the separation method will con-
dition the isolation/extraction methods.
2.1.1.1 Sample collection and processing
At first, sample collection should be controlled as strictly as
possible. For instance, tissue samples should include when
possible, biopsies from both unaffected and affected regions
as well as a representative distribution of certain parameters
such as gender [7], type [7, 8]. In addition, sample collection
must be well documented with as much clinically relevant
information as possible. When cultured cells are used, highly
consistent culture conditions are required. As for any
experimental approach, sample preparation is the most crit-
ical part of proteomics experiments. This step involves tis-
sue/cell homogenization and/or lysis. However, conditions
that maintain protein solubility and are compatible with the
downstream methods are frequently the most difficult to
achieve. For instance, the nature of detergents used for
membrane solubilization as well as the presence of specific
ions (Mg21 and Ca21) may be of importance relative to the
methods selected for protein separation. Similarly, inhibition
of proteases and phosphatases is critical for accurate analy-
ses.
2.1.1.2 Sample prefractionation
In several cases, sample fractionation may be necessary either
to reduce sample complexity and/or to enrich in a particular
sample subset. Typical examples are the isolation of cellular
compartments or specific molecular machines. Subcellular
proteomics is now a well-accepted methodology to determine
the content of cellular compartment. These approaches have
led to significant successes [9, 10] and revealed novel and
unexpected cellular functions [11]. Besides subcellular frac-
tionation, the isolation of specific molecular machines such
as ribosomes, proteasome, or other large subcellular com-
plexes [12] may provide significant enrichment of their com-
ponents for MS analyses. Traditionally, subcellular fractiona-
tion has been accomplished after cell/tissue homogenization
by physical methods such as density gradient centrifugation,
but immunomagnetic separations are also used for more
highly purified subcellular fractions [13]. Finally, in a number
of cases, and more particularly for serum proteomics, various
steps of depletion have been established in an attempt to
reduce the amount of very abundant proteins such as albu-
min and as a consequence decrease the dynamic range of the
analysis [14, 15].
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Systems Biology 5469
2.1.1.3 Protein separation
With or without sample prefractionation, the isolated pro-
teins still represent a too highly complex mixture for being
directly analyzed by MS. Therefore, a protein separation step
is applied in most of the proteomics workflow. Two major
protein separation approaches can be undertaken using
either gel-based or nongel-based methods. In addition, a
combination of both approaches can also be considered. Gel-
based methods include both 1-DE and 2-DE. Although 1-DE
still represents a significant separation tool to resolve pro-
teins, proteomics is closely associated with 2-DE, because of
the separation power and quantification provided by staining
and image analysis. However, there are a number of reasons
that 2-D gels are less than optimal for proteomics studies
such as poor routine separations at extremes of molecular
weight (MW) (limited from 10 to 150 kDa) or pI (limited
from 3 to 10), LOD for staining (ng), limited dynamic range
for staining (below 1:1000), solubility problems for some
integral membrane proteins, and labor-intensive procedures.
Although many of these limitations are being addressed,
such as membrane protein separation [16] and dynamic
range, routine 2-D gels do not represent a universal separa-
tion technique for all proteins [17, 18]. Alternatively, protein
chromatography represents a very efficient strategy to sepa-
rate intact proteins preproteolysis. Interestingly, a recent
study carried out on serum samples revealed that when the
aim of the analyses is to detect as many proteins as possible,
the use of protein prefractionation methods coupled with
multidimensional protein identification technology (Mud-
PIT; [19]) was the most effective in identifying many proteins
and in a better coverage of individual proteins. This approach
included strong cation exchange (SCX) chromatography on
intact proteins followed by trypsin digestions, SCX chroma-
tography, RP-HPLC and ESI-MS [20, 21]. Additionally, this
type of protein fractionation prior to MS analyses is particu-
larly suitable for membrane proteins, however, in this case
an additional separation step such as 1-DE could also be car-
ried out [22].
2.1.1.4 Sample preparation for MS analyses
In bottom up analyses, proteins are digested with proteoly-
tic enzymes such as trypsin (or Glu C, etc.) prior to MS se-
quencing. These methodologies have been extensively
described in previous studies and are well accepted.
Another aspect of sample preparation pertains to protein
quantitation by either labeling intact proteins prior to pep-
tide generation (stable isotope labeling with amino acids in
cell culture, SILAC) [23, 24] or peptides resulting from
trypsin digestion (isotope-coded affinity tags; ICAT or iso-
baric tags for relative and absolute quantitation; iTRAQ)
[25–28]. These approaches allow for the relative comparison
of two biological samples (e.g., normal vs. disease or control
vs. treated). Indeed, in the case of SILAC, stable isotopes are
incorporated into proteins prior to isolation. This occurs by
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5470 S. Palcy and E. Chevet
selective metabolic labeling with 15N containing amino
acids (such as leucine). In ICAT technique, labeling is per-
formed after protein isolation through alkylation of cysteine
groups with either an isotopically heavy reagent that incor-
porates eight deuterium atoms in place of eight hydrogen
atoms (i.e., d8-ICAT) or a light reagent containing the natu-
ral distribution of elements (i.e., d0-ICAT). After labeling,
protein samples are combined and digested with trypsin.
Peptides are fractionated by SCX chromatography and fur-
ther resolved by microcapillary RP LC, before MS analysis
using IT, hybrid quadrupole TOF (Q-TOF), or TOF–TOF
instruments. Because isotopically labeled peptide pairs are
biochemically identical, they coelute during column chro-
matography [27].
2.1.2 MS sequencing and analyses
At this stage of the approach several options are yet possible.
PMF by MALDI-TOF MS of peptide mixtures has the ad-
vantage of being very sensitive, automatable for high-
throughput measures and very accurate for mass determi-
nations. It has been shown that very small amounts of the
proteolytic digest are sufficient to provide an excellent
MALDI spectrum [29–31]. In the measurement of a tryptic
peptide digest, intense peaks from trypsin autoproteolytic
fragments may be used as internal standards. In addition,
internal peptide standards may also be added to achieve
greater mass accuracy. When the peptide mass mapping
experiments do not yield unequivocal matches, fragment
ions measurement by MS/MS or PSD are carried out. The
availability of tandem mass spectrometers with MALDI
sources, such as Q-TOF [32, 33] and TOF–TOF [34], also
permits the acquisition of more extensive fragmentation for
one or more peptides to confirm the peptide mass map
results.
Generally, peptide fragmentation spectra are obtained
by ESI-MS/MS. Triple quadrupole, quadrupole IT, and Q-
TOF are the typical mass analyzers. The fragment ion spec-
tra are acquired in a data-dependent manner (i.e., the pres-
ence of a peptide mass above a specified intensity threshold
triggers the automated generation of the fragment ion
spectrum for that precursor mass). A comparative study of
nanospray versus LC-MS/MS showed that in a complex
mixture fragment ion spectra could be generated for a
greater number of peptides by use of LC-MS/MS rather
than nanospray [35]. The use of LC-MS/MS for complex
peptide mixtures has been enhanced in a number of ways.
Peptide separation and identification can be enhanced fur-
ther by increasing the ability of the mass spectrometer to
perform MS/MS on every component. Normally the tran-
sient nature of components eluting from LC columns limits
the number of coeluting peptides that can be selected and
fragmented by MS/MS. For peptide relative quantitation,
the mass spectrometer toggles between survey scans (MS),
and sequencing scans (MS/MS). The ratio of ion intensities
from coeluting labeled peptides is determined in the survey
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 5467–5480
scan and defines the ratio between parent proteins in the
starting samples. MS database searching will be discussed
in Section 3.
2.2 Reverse proteomics
Whereas in classical proteomics, the starting material is
isolated proteins which are analyzed via MS techniques, and
identified using complete genome sequences, in reverse
proteomics, the starting point is the genome sequence of an
organism. First, the transcriptome and proteome are pre-
dicted in silico and subsequently this information is used to
generate reagents for gene functional analysis. This experi-
mental strategy includes several steps such as cDNA clon-
ing, protein expression (several systems from Escherichia
coli to mammals are available) and specific functional assays
based on gene function annotations. When reverse prote-
omics is applied downstream of a forward proteomics pipe-
line, the experimental design will be focused on the genome
subset corresponding to the previously identified gene
products.
2.2.1 Cloning
Historically, expression clone collections have relied on
cDNA libraries cloned as a pool into specific vectors. To
generate such libraries, mRNA collected from an appro-
priate source is reverse-transcribed to DNA, and then
introduced into a vector. Complementary DNA libraries
have found considerable success in the study of prokar-
yotes and simple eukaryotes, but several issues such as the
presence of the 5’ and 3’ untranslated regions have limited
their application for genetic studies in more complex spe-
cies (metazoan). In addition, the translational reading
frame of the tag with respect to the coding sequence is not
known, and the UTR sequences themselves may contain
in-frame stop codons. Therefore, it becomes of interest to
create clones that contain only coding sequences, often
referred to as ORF clones. Typically, four types of starting
material may be used as template to generate clones:
(i) genomic DNA; (ii) first-strand cDNA; (iii) cDNA library;
or (iv) pre-existing sequence validated full-length cDNA
clones (such as those available from the Mammalian Gene
Collection (MGC; [36–38]) or RIKEN [39, 40]. Genomic
DNA is the best template, because all genes are equally
represented, but it is only applicable to organisms with lit-
tle or no splicing, limiting it to prokaryotes and simple
eukaryotes.
Nevertheless, the challenges associated with ORF clones
creation are clearly outweighed by the advantages of their
use, such as the opportunity to execute parallel experiments,
in which all or a subset of genes in a genome can be tested in
a controlled setting under identical conditions. Moreover,
since their identity is known in advance, information
regarding all clones can be recorded.
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Proteomics 2006, 6, 5467–5480
To date, besides the earliest comprehensive (ORF) clone
collections which were constructed with gap-repair cloning, a
method of cloning that uses homologous recombination in
the yeast Saccharomyces cerevisiae [41–43], the best example of
reverse proteomics cloning platform for metazoan genomes
was provided by the work initiated by the Vidal lab [44–46]
where ORFs were deduced from the analyses of complete
genome sequences. This was first demonstrated by the gen-
eration of the Caenorhabditis elegans ORFeome [47] and since
then a significant number of similar projects have followed
including the Arabidopsis thaliana, Sinorhizobium meliloti,
Brucella melitensis, and Human ORFeomes [48–51]. These
ORF collections can now be used in the assays described in
Section 2.2.2.
2.2.2 Expression and functional assays
These experimental systems will provide the basic recombi-
nant material to conduct further protein characterization
studies including protein functional assays (see Fig. 2).
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Systems Biology 5471
2.2.2.1 Expression systems
These experiments can be carried out in vitro or in vivo
including bacterial to mammalian expression systems. The
expression of recombinant protein can serve several pur-
poses such as antibody production, structural, or functional
studies. At this stage, the nature of the protein (size, presence
of transmembrane domains, acidic or basic stretches, and
proteins normally secreted) and the application chosen must
be considered for experimental design including the choice
of the expression system. For instance, in the case of anti-
body production or structural studies, significant amount of
recombinant proteins from specific ORFs must be gener-
ated. Furthermore, proteins containing transmembrane
regions will most likely be insoluble in bacterial expression
systems. This type of problem can be solved by selecting
eukaryotic expression systems or expressing protein frag-
ments/domains, the latter representing a simpler solution.
Similarly for structural genomics projects, protein solubility
and folding are of course of major interest [52].
Figure 2. Schematic repre-
sentation of the reverse prote-
omics workflow.
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5472 S. Palcy and E. Chevet
Today, there is a large variety of expression systems
available for recombinant protein production, each having its
own respective advantages in relation to cost, ease of use, and
their PTM profiles. (1) Traditional cell-free expression sys-
tems have always been limited by low expression levels.
However, the use of a eukaryotic based approach coupled
with the rapid, cell-free utility offered by the system has a
number of clear advantages and offers a viable alternate to
traditional approaches [53]. (2) Bacterial expression is prob-
ably the most commonly used expression system for the
production of recombinant proteins. However, since it is a
prokaryotic based system, heterologously expressed eukar-
yotic proteins are not modified correctly. Furthermore, pro-
teins expressed in large amounts can precipitate, forming
inclusion bodies, and large complex proteins may also be
difficult to propagate. (3) Baculovirus-mediated insect cell
expression has become one of the most popular vehicles for
the production of large quantities of recombinant protein for
structural and functional studies. Baculovirus protein
expression is a eukaryotic based expression system and thus
offers protein modification and processing patterns similar
to those in higher eukaryotic cells. However, a major limita-
tion of the baculovirus expression system has always been
that the N-glycosylation pathway of insect cells, specifically
those of Sf9 and Sf21 (the most commonly used cell lines) is
different from that found in mammalian cells [52]. (4) Yeast,
in particular Pichia pastoris, offers a very powerful option for
secretory proteins, and offers a very powerful alternative to
baculovirus or mammalian expression systems, specifically
in regard to the generation of large quantities of secreted
material [52]. (5) Finally, the expression of proteins in a
mammalian background offers many clear advantages to
their generation in E. coli or insect cells, including correct
PTM and folding. However, although the use of mammalian
cells such as CHO or HEK293 is well documented, the pro-
cess of creating stable mammalian cell lines can often be la-
borious and time consuming [52].
2.2.2.2 Functional assays
These assays will contribute to the functional characteriza-
tion of gene products. This information can be integrated at
genome-wide levels and provide therefore functionally rele-
vant annotation of the gene products studied.
Subcellular localization: Determination of the subcellular
localization and dynamics of proteins is a very important first
step toward the understanding of their cellular function. For
instance, proteins of a given functional network must colo-
calize in order to interact with each other, or protein relocal-
ization in response to external stimuli may reflect changes in
activities. Systematic experimental pipelines to determine
the subcellular localization of proteins have been set in place
in various systems. The most comprehensive studies have
been carried out in yeast where systematic tagging of the
endogenous ORF was performed with GFP [54, 55]. In addi-
tion, in mammalian systems several initiatives have also
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 5467–5480
been undertaken (http://www.dkfz.de/LIFEdb; [56–58]) by
systematic tagging of specific ORFs with fluorescent protein
encoding sequences and transfected in various cell lines. A
major limitation of these projects is the presence of the GFP
tag on the original protein sequence which can lead to pro-
tein mis-localization. An alternative to this approach is pro-
vided by the systematic use of antibodies as illustrated by the
protein atlas project ([59–61]; www.proteinatlas.org).
Protein–protein interactions: The characterization of pro-
tein function by the identification of specific functional part-
ners has been tackled by the establishment of protein–pro-
tein interaction networks at a genome-wide level. The use of
yeast two-hybrid has allowed the establishment of the first
interaction map of a complete organism S. cerevisiae in 2001
[62], the evolution of this technology has then allowed the
establishment of genome-wide protein–protein interaction
maps in C. elegans [63], Drosophila melanogaster [64, 65], more
recently in human [66] and soon in mouse. Similarly,
approaches using MS were used to identify and characterize
protein complexes. These strategies involve the use of tan-
dem affinity purification (TAP) tags [67] which allows the
elimination of a vast majority of contaminant usually found
in classical immunoprecipitations studies. Comprehensive
genome-wide protein–protein interaction maps were gener-
ated in yeast using this approach [68] as well as several others
in mammalian systems but at lower scales [69].
Enzymatic activities: The systematic analysis of enzymatic
activities at a genome scale requires the development of
enzymatic assays allowing for high-throughput studies.
Thus far, such assays have been developed mainly to screen
for compounds altering the activity of a given protein/en-
zyme [70–72]. However, in the case of reverse proteomics, the
objective would be to systematically identify substrates for a
family of enzymes which implicates on the one hand
recombinant protein expression systems able to handle the
expression of a large spectrum of proteins with not necessa-
rily large quantities (mg). Such studies have been carried out
in S. cerevisiae where the yeast kinome was studied using a
protein chip technology [73], in addition several other assays
have also been developed to allow high-throughput studies of
small [74] or trimeric G proteins [72]. Therefore, the devel-
opment of assays allowing for the analysis of enzymatic ac-
tivities (either intrinsic properties or substrate identification)
represents another step in the characterization of proteins in
a reverse proteomics pipeline.
Gene silencing: These approaches contribute importantly
to the understanding of molecular functions related to spe-
cific phenotypes. These studies initially performed using
gene deletion strategies in various organisms have more
recently included RNA interference approaches. Specific
single or double gene knockouts have been carried out in a
systematic fashion in S. cerevisiae and the impact of these
deletions on cell survival [75, 76] and other specific pheno-
types evaluated. Similarly, RNA interference has become in
the past 5–10 years a major technology in functional geno-
mics providing specific gene silencing and upscalable to
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Proteomics 2006, 6, 5467–5480
high-throughput [77]. The first genome-wide approaches
were carried out on the worm C. elegans [78]. Since then these
high-throughput approaches have been extended to other
systems including D. melanogaster or mammalian cells in
culture [79–83]. Analyzed in the context of specific pheno-
types (which can be molecular as well), these systematic
approaches have provided numerous functional information
on the genes studied.
Integration of the above described datasets to unravel protein
function: Several examples are now available where either at
the level of a single protein or at the level of a large number
of proteins, the integration of datasets providing hetero-
genous information has allowed the identification of protein
functions. For instance, this was the case for a small GTP
binding protein of the Ras superfamily identified by
sequence homology in the C. elegans genome [84]. By col-
lecting information on its subcellular localization, its tis-
sular distribution in the worm and its activity, it was made
possible to postulate a potential biological role in the main-
tenance of the apical polarity in epithelial cells. Similarly
but at a much larger scale, Gunsalus et al. [85] reported the
establishment of phenoclusters in the worm based on the
integration of heterogenous datasets. This process has
allowed the understanding of C. elegans early embryogen-
esis at a system level.
2.3 Integration of forward and reverse experimental
pipelines
Individually, both forward and reverse proteomics are two
approaches with very high discovery potential for protein
characterization. However, we can postulate that the sys-
tematic integration of both strategies (see Fig. 3) may repre-
sent an additional advance in the understanding of protein
functions in a specific biological context. To integrate these
two experimental pipelines as described in Fig. 3, several
parameters must be considered including a very strict defi-
nition of the initial biological sample, and a data integration
pipeline between both forward and reverse steps. A very
elegant example of such an experimental strategy has been
recently provided by Skop et al. [86], who purified mamma-
Figure 3. Integration of forward and reverse proteomics.
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Systems Biology 5473
lian midbodies, identified the components using a forward
proteomics approach and tested the functionality of these
components in a reverse pipeline using C. elegans as a
model system thus revealing conserved cytokinesis mech-
anisms.
The definition of the biological sample must include
information about its nature (model and experimental con-
ditions). Additional information about similar samples from
either the literature or publicly available datasets must be
collected and used for initial sample annotation. Then, the
list of proteins resulting from MS analyses will constitute the
final information to condition the reverse approach. Al-
though, the forward workflow is not affected in such inte-
grated pipeline, the strategy for collecting and storing the
information must be applicable for a downstream integra-
tion with the reverse step. The actual constraint is therefore
applied to the reverse pipeline. Indeed, the limitation is no
longer imposed by the identification of ORFs in a given ge-
nome but by the list of proteins identified in the forward
approach. This list must be analyzed (see Section 3) to pro-
vide sequence feature information (e.g., presence of trans-
membrane domains) and eventually functional annotation.
In addition, the nature of the initial biological sample will
also condition the nature of the assays which will be under-
taken for the functional evaluation of the identified compo-
nents. Finally, the data harvested through the reverse pipe-
line will complement the initial knowledge collected on the
biological sample and lead to comprehensive analysis of the
system studied.
3 Data analysis and integration
Forward and reverse proteomics analyses cannot be per-
formed without the assistance of bioinformatics resources
for data harvesting, management, storage, and analysis.
Throughout all the steps, bioinformatics will support the
analytical workflow and will provide the appropriate inter-
face for the interpretation of the data in a biologically relevant
context.
3.1 Protein identification
Protein identification via MS is achieved using searching
against molecular sequence databases (see Table 1). In brief,
peptide or peptide fragment masses are matched to theoret-
ical masses predicted from in silico digested protein sequen-
ces [87]. Three major criteria have to be considered when
undertaking protein identification using MS data: (i) the
quality of the protein identification (as indicated by match
scoring and protein sequence coverage), (ii) the total number
of identified proteins, and (iii) the percentage of false posi-
tive. These criteria are not only influenced by the quality of
the MS data acquired but also by the proficiency of the
bioinformatics resources.
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5474 S. Palcy and E. Chevet Proteomics 2006, 6, 5467–5480

Table 1. Molecular sequence databases

The International Nucleotide Sequence Database Collaboration (INSDC), http://www.insdc.org/

Name Database features Description Number of total
(or selected) entries

Nucleotide and genome databases

GenBank (NCBI) General This database contains a collection of publicly available DNA sequences for 54 584 635
Comprehensive more than 205 000 organisms. The sequence information is obtained from February 06
Minimal annotation individual laboratories and batch submissions from large-scale
Redundant sequencing projects. The records include annotation using a standard
Noncurated set of biological terms and crosslinks to taxonomy, genome, mapping,
protein structure, and domain information as well as the literature
database PubMed.
URL: http://www.ncbi.nlm.nih.gov/
dbEST (NCBI) General dbEST is the division of GenBank that contains “single-pass” cDNA 32 889 225
Minimal annotation sequences, or ESTs, from a number of organisms. January 06
Redundant URL: http://www.ncbi.nlm.nih.gov/
Noncurated
EMBL (EBI) General The EMBL Nucleotide Sequence Database contains DNA and RNA 64 739 883
Comprehensive sequences collected from individual researchers, genome sequencing December 05
Minimal annotation projects, patent applications and more.
Redundant The database is highly integrated with other data resources offered at
Noncurated the EBI and elsewhere (URL: http://www.ebi.ac.uk/embl/)
DDBJ DNA (Data Bank General This database contains DNA sequences including cDNAs, ESTs, genomic 52 272 669
of Japan) Comprehensive DNAs, sequence tagged sites (STS), and synthetic constructs December 05
Minimal annotation (URL: http://www.ddbj.nig.ac.jp)
Redundant
Noncurated
Genomic databases
NCBI Entrez Genome General This general genomic resource includes genomes of over 1000 organisms. 5202
Comprehensive Complete and partial genomes are included March 06
Moderate annotation (URL: http://www.ncbi.nlm.nih.gov/)
Redundant
Noncurated
MGD (The Jackson Specific The Mouse Genome Database is a highly curated and annotated resource 30 927 (genes)
Laboratory) Comprehensive on mouse genome which presents an integrated view of genotype March 06
Extensive annotation (sequence) to phenotype information including genes and gene
Nonredundant products. MGD includes homology data for mammalian orthologs,
Curated alleles and targeted mutation reports, and more
(http://www.informatics.jax.org)

Protein databases
NCBI Entrez Protein General The database is a compilation of translated nucleotide sequences from the 8 212 494
Comprehensive annotated coding regions in GenBank and RefSeq as well as protein March 06
Moderateannotation entries from Swiss-Prot, Protein Information Resource (PIR), Protein
Redundant Research Foundation (PRF), and Protein Data Bank (PDB). Protein
Noncurated sequence records in Entrez are linked to the other NCBI resources such
as precomputed protein BLAST alignments, protein structures,
conserved protein domains, nucleotide sequences, genomes, and genes
(URL: http://www.ncbi.nlm.nih.gov/entrez)
NCBInr General Nr is a composite database which contains all nonredundant GenBank 3 479 934
Comprehensive coding sequence translations, RefSeq Proteins, PDB, Swiss-Prot, PIR, March 06
Moderate annotation and PRF entries. The sequences in nr are crossreferenced to their
Nonredundant initial databases in order to avoid duplication
Noncurated (URL: http://www.ncbi.nlm.nih.gov)
RefSeq Protein (NCBI) General The database provides reference proteins sequence. Records are compiled 2 273 764
Moderate annotation using a combined approach of collaboration, automated methods, January 06
Nonredundant prediction, and curation. They are highly integrated with other NCBI
curated resources (URL: http://www.ncbi.nlm.nih.gov/RefSeq)

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Proteomics 2006, 6, 5467–5480 Systems Biology 5475

Table 1. Continued

The International Nucleotide Sequence Database Collaboration (INSDC), http://www.insdc.org/

Name Database features Description Number of total
(or selected) entries

Swiss-Prot (Swiss-Prot General Swiss-Prot Protein Knowledgebase is a manually curated protein sequence 211 104
group and SIB) Extensive annotation database which provides a high level of annotation (including protein March 06
Nonredundant function, domains structure, PTMs, and variants). All sequence reports
curated corresponding to the same gene product are merged into a single entry
which is currently crossreferenced with about 60 different databases.
Protein records are annotated and reviewed by biologists to ensure that
the database is of a high quality (URL: http://www.expasy.org/sprot)
TrEMBL (Swiss-Prot General This database is a computer-annotated supplement of Swiss-Prot that 2 638 494
group and SIB) Extensive annotation contains all the translations of INSDC entries, not yet integrated in March 06
Nonredundant Swiss-Prot. Besides, the collection includes protein sequences extracted
curated from the literature or direct submission. As in Swiss-Prot, information
redundancy is avoided by merging several sequence records of the same
gene product. Together, Swiss-Prot and TrEMBL constitute a
comprehensive resource for all the publicly available protein sequences
(URL: http://ca.expasy.org/sprot/)
PIR-PSD (Georgetown General The Protein Information Resource Protein Sequence Database, the world’s 283 009
University Medical Comprehensive first database of classified and functionally annotated protein sequences. December 2004
Center) Extensive annotation The resource compiles protein sequences, organized by superfamily and
Nonredundant family, and annotated with functional, structural, bibliographic, and
curated genetic data. As all PIR-PSD entries has been merged into the UniProt
databases, it is no longer updated (last release in December 2004,
URL: http://pir.georgetown.edu/pirwww/dbinfo/pir_psd.shtml).
The database is extensively crossreferenced with INSDC collection,
PubMed and MEDLINE IDs, and many other databases
UniProt (UniProt General The Universal Protein Resource is the most comprehensive collection of 2 812 716
Consortium-EBI, SIB, Comprehensive protein sequences today. It is a joined resource of protein sequence February 06
and PIR) Extensive annotation repository and functional information. The resources include three
Nonredundant components: (i) UniProt Knowledgebase (UniProtKB) in which the protein
Curated sequence and functional information from Swiss-Prot, TrEMBL, and
PIR-PSD entries are merged, (ii) UniProt Reference Clusters (UniRef)
which provides a nonredundant view of the resources using several level
of sequence identity and (iii) UniProt Archive (UniParc), the most
comprehensive and nonredundant collection of publicly accessible
protein sequences from Swiss-Prot, TrEMBL, PIR-PSD, EMBL, Ensembl,
International Protein Index (IPI), PDB, RefSeq, FlyBase, WormBase and
the patent offices in Europe, the United States, and Japan
(URL: http://www.pir.uniprot.org/)
Example of specific protein databases
Brenda (University Specific BRENDA is a comprehensive resource on functional and molecular 3996 (EC numbers)
of Cologne) Comprehensive information of enzymes, based on primary literature. It contains all January 04
Extensive annotation enzymes classified according to the EC system of the Enzyme
Nonredundant Nomenclature Committee (IUBMB). The database content include
Curated enzyme sequence, structure, specificity, stability, reaction parameters,
and isolation data (URL: http://www.brenda.uni-koeln.de)
Histone Sequence Specific This database is a collection of histone-fold containing sequences derived 1378 (unique
Database (NHGRI/ Comprehensive from sequence-similarity searches of public databases. Sequence sets sequences, all
NCBI) Minimal annotation are presented in redundant and nonredundant, linked to GenBank histone classes)
Curated sequence files. For each class of eukaryotic histone, sequence- and January 05
text-based queries have been used to extract all relevant entries from
NCBIs Entrez protein sequence database
(URL: http://genome.nhgri.nih.gov/histones/)

Sequence databases are designed for the direct archiving of experimental results. They are termed as primary databases and contain
more or less associated information such as literature references, functional annotation and crossreferences to other sequence data-
bases.

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5476 S. Palcy and E. Chevet
3.1.1 Molecular sequence databases
The choice of the database is an important factor which can
influence protein identification. Several aspects of the data-
base have to be taken into consideration: (i) the source of the
information, (ii) the nature and specificity of the informa-
tion, and (iii) the size of the database. In protein databases,
the sequence information may have several sources. Some
proteins sequences result from Edman degradation of puri-
fied gene products. Others are sequences from functional
cDNA cloning and mRNA characterization. Finally, a signif-
icant part corresponds to putative protein sequences deduced
from genome annotation and EST databases. The degree of
uncertainty regarding the exact protein coding sequence
varies among these different sources. Indeed, information
on the size of the gene product, translation initiation start,
reading frame, and occurrence of alternative splicing could
be more or less accurate [88]. To compensate the lack of con-
fidence toward certain sources, sequence validation strate-
gies have been put in place to automatically [89] or manually
validate the available information [90].
Molecular sequence databases can be classified as either
“general” or “specific”. The general databases contain all
types of protein sequence entry from several organisms/
species, such as Swiss-Prot, TrEMBL, Refseq (NCBI), and
PIR [91]. Besides, specific databases contain protein entries
relative to an organism/species, a category of protein (such
as enzymes) or a protein superfamily (such as histones) (see
Table 1). Furthermore, a clear distinction has to be made be-
tween databases which are only sequence repositories (e.g.,
Genbank and TrEMBL) and annotated/curated databases
(e.g., Pir, Swiss-prot, and RefSeq). Finally, a new generation
of databases, named “metadatabases” is a database of data-
bases which groups under a unique entry the information
found in several resources (e.g., UniProt).
Most of the global approaches to forward proteomics
use general protein databases for protein sequence identifi-
cation, with or without species restriction, depending on
genome data availability [92, 93]. This is due to the fact that
the comprehensiveness of the selected databases is critical
for guarantying a high number of protein identification.
However, the direct use of sequence repositories will be
avoided due to their high level of redundancy [91]. Database
redundancy is a real problem which affects the searching
time. Moreover, the amino acid distribution in the database
will also be different and this will impact on some scoring
models [94]. Nevertheless, sequence repository databases
allow the rapid availability of sequence information to the
community, and when an appropriate filter is applied to
decrease the information redundancy, these refined sources
of protein sequences become a powerful tool to be used for
protein identification (e.g., NCBInr). When undertaking a
more specific project, the use of curated protein databases is
of great interest. Indeed, in addition to an absence of
redundant sequences, these resources present the advan-
tages to be well annotated and crossreferenced to other
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2006, 6, 5467–5480
databases such as 3-D structures, 2-D gels, protein–protein
interactions, protein families, or literature databases.
Searches using genomic or EST databases can identify
additional protein species which have not yet been incorpo-
rated in protein databases [95]. However, this information
source of data presents several challenges. First, nucleic acid
sequences require the translation into the six possible read-
ing frames prior to be used for searching with mass spectra.
This extends considerably the time allocated for searches.
Second, genomic data, in the case of eukaryotes, contain a
mixture of introns and exons, and a peptide sequence may
overlap two different exons which will therefore prevent its
identification due to the lack of a contiguous sequence in the
genomics database [96].
When using large databases, the impact on the identifi-
cation (positive match) and its quality (e-value), must be
taken into consideration. The size of the database can indeed
affect the positive identification of unique peptides in shot
gun approaches [97].
A general issue with the use of any molecular sequence
database for peptide searching is that none of the PTMs
encountered in the biological context or chemical modifica-
tions occurring during the protein separation will be taken
into account. While chemical modifications due to the
experimental processes are well identified [98] and therefore
easily predictable, the nature and number of the PTMs
represent a real challenge for global approaches. Therefore,
specific analytical workflows (using methods of sample iso-
lation and/or MS analysis, see Section 2) should be applied
for PTMs detection at a large-scale [99]. Otherwise, it would
be extremely difficult to identify the modified peptides due to
poor abundance and limitation in the database searching. As
a consequence, a list of unassigned and incorrectly assigned
peptides would be returned. This is a major concern of the
field that some groups have attempted to address [100, 101].
3.1.2 MS and MS/MS database searching
Comparison of MS data to in silico digested protein database
is performed using bioinformatics tools commonly named
“MS search engines”. Several algorithms for peptide identi-
fication are used in forward proteomics approaches such as
SEQUEST, MASCOT, and ProteinProspector [94]. The prin-
ciple of these algorithms is to match molecular mass values
of peptides or peptide fragments to theoretical mass values
predicted from protein sequence databases. When several
peptides match the same protein entry, they are clustered
together. The protein entry is then reported as a protein hit.
Two main methodologies have been developed for pro-
tein identification using MS (see Section 2): (i) PMF (using
MS data) and (ii) peptide mass tag (using MS/MS data). The
choice of one or the other method is determined by the pro-
tein sample complexity and the database to be searched.
PMF cannot be applied when the protein mixture is too
complex or when searching against a genomic or EST data-
base [95]. Similarly, the choice of the MS search engine will
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Proteomics 2006, 6, 5467–5480
influence the list of the validated peptide matches. All search
engines will return peptide matches based on the criteria set
up by the investigator such as database used, mass tolerance
(according to the mass accuracy of the mass spectrometer
used), ion charge state, chemical (depending on protein
separation methods) and PTMs, number of proteolytic mis-
cleavages, etc. However, the validity of the peptide matches
and corresponding protein hits is controlled by a scoring
model which varies among the different search algorithms.
The quality of the search is then defined by the number of
false positive and unassigned MS spectra. Clearly, the choice
of the MS search engine is a determinant for the success of a
proteomics analysis and already approaches have been
developed which combine results from different search
engines for confirmation of peptide assignment and higher
comprehensiveness [102].
3.2 Protein annotation
Protein annotation provides the basic elements for under-
standing the biological relevance and context of the identified
proteins. The proteins are annotated with information
derived either from experimental data or prediction via
sequence similarities [103]. Annotations of protein provides
information ranging from protein sequence features to elu-
cidated biological function(s) and partner(s), including tissue
distribution, subcellular localization, and involvement in
pathologies. In the context of forward proteomics, where the
datasets generated are protein sequences, this step mainly
implies the annotation of the primary structure. A large
panel of bioinformatics tools is now available to analyze
sequence homologies, detect conserved domains, motifs,
and signatures as well as predict topology, subcellular locali-
zation, and 3-D structures. The analysis of protein sequence
homologies using sequence global alignment tools such as
CLUSTALW [104] or MULTALIN [105] returns extremely
useful information about gene product conservation among
species and the occurrence of a close homolog. The detection
of a high degree of homology with well-characterized pro-
teins provides a good link to the putative function of
unknown proteins [106]. Furthermore, the use of local
alignment tools such as BLAST [107], PSI-BLAST [108], or
SIM [109] informs on domain and pattern homology. This
may also highlight new conserved domain or signatures and
as such uncover new protein families.
Further information to predict protein function can also
be obtained by scanning protein sequences against a
sequence feature database such as Pfam, Prosite, Smart, and
Blocks. The metadatabase InterPro (an excellent resource of
integrated documentation), unites the information of
11 resources for protein sequence features [110] such as
protein families, conserved domains, and functional sites. Its
associated tool, InterProScan [111], scans sequences against
the InterPro database members and returns an integrated
protein sequence analysis. Prediction of transmembrane do-
main and topology (Psort [112] and TMHMM [113]) is a crit-
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Systems Biology 5477
ical element of protein functions and is of high importance
for the design of subsequent experiments in the reverse pro-
teomics phase. Similarly, the prediction of subcellular locali-
zation (Psort and SignalP [114]) could also bring additional
elements on protein function and how it should be assessed
experimentally. For all the tools mentioned above, the amino
acid sequence is sufficient to obtain some information.
However, the prediction of 3-D structure from protein
sequence is a hardest task to achieve. Nevertheless, model
prediction can be obtained with knowledge-based algorithms
such as ProMod [115]. The comparative modeling method
allows the prediction of 3-D structures of proteins sharing
.30% of sequence identity with a protein with a known 3-D
structure [116]. For well-characterized proteins, reliable
annotation information can simply be retrieved from curated
databases such as UniProt [117].
One important aspect of protein annotation is the use of
controlled vocabulary, otherwise the use of the information
would lack homogeneity and crossdatabase searches would
become extremely difficult. The Gene Ontologies con-
sortium has produced the first and the mostly used con-
trolled vocabulary index in genomics [118]. The GO database
has become an essential annotation resource for describing
the roles of genes and gene products in any organism. The
GO terms are used by several collaborating databases which
facilitates automatic transfers of annotation. Recently, GO
Engine, a computational platform for GO annotation have
been developed and used for large-scale annotation of hu-
man proteins [119].
3.3 Organizing the data
Forward proteomics analyses return such large amount of
data that their direct interpretation or utilization in further
analytical studies is extremely difficult. To overcome this
problem, data clustering methods can be applied in order to
turn these large datasets into manageable and meaningful
subsets. Indeed, using the annotation information, proteins
identified through the forward step can then be classified
based on similar attributes (sequence, localization, function,
etc.).
When combining forward and reverse proteomics
approaches, it becomes of high importance to partition the
identified proteins into functional subsets or clusters which
will provide the framework for the design and selection of
the subsequent functional assays. As for the annotation step,
the clustering steps cannot be undertaken manually when
the scale of the analysis is too large and automated processes
have to be implemented [120].
3.4 Data integration
In the forward–reverse proteomics pipeline, the genes cor-
responding to the proteins identified via MS analysis (for-
ward step) will be then subjected to functional analysis
(reverse step). Due to crossreferences between protein and
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5478 S. Palcy and E. Chevet
genome database, DNA sequence information is easy to
access and in most cases, is linked to either gene collections
or ORFeomes. Therefore, the corresponding DNA material
will be either simply retrieved from DNA collections or
cloned. Once expressed, the subsequent recombinant pro-
teins will be tested according to their functional annotation.
The new set of data collected through the reverse prote-
omics analysis will expend the knowledge on the proteins.
This information will therefore constitute a new source of
protein annotation which could be used for another run of
either reverse proteomics analysis or setting new analytical
condition for the forward proteomics workflow.
4 Conclusions
The integration of the data collected in a linked fashion [121],
using systems which allow for querying and navigating
multiple databanks, or crossreferences between entries from
different databases, and those generated experimentally
represents the next challenge to complete a system repre-
sentation of the biological model studied. As a consequence,
systems biology type of integration should be based on
modeling the initial biological system by integrating infor-
mation collected at the level of each of its individual compo-
nents thus revealing new knowledge through the emergent
properties of the model.
We apologize to those whose work was not cited in this review
due to space limitation. We thank the Chevet lab for critically
reviewing this manuscript. E. C. is a Junior Scholar from the
Fonds de la recherché en Santé du Québec (FRSQ).
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