1478 DOI 10.1002/pmic.

201100563 Proteomics 2012, 12, 1478–1498

REVIEW

History of protein–protein interactions: From egg-white

to complex networks
Pascal Braun1,2 and Anne-Claude Gingras3,4
1
Department of Plant Systems Biology, Center for Life and Food Sciences Weihenstephan, Technical University
Munich, Freising, Germany
2
Research Unit Protein Science, Helmholtz Centre Munich, Munich, Germany
3
Samuel Lunenfeld Research Institute at Mount Sinai Hospital, Toronto, Ontario, Canada
4
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

Today, it is widely appreciated that protein–protein interactions play a fundamental role in Received: October 27, 2011
biological processes. This was not always the case. The study of protein interactions started Revised: January 4, 2012
Accepted: January 17, 2012
slowly and evolved considerably, together with conceptual and technological progress in differ-
ent areas of research through the late 19th and the 20th centuries. In this review, we present
some of the key experiments that have introduced major conceptual advances in biochemistry
and molecular biology, and review technological breakthroughs that have paved the way for
today’s systems-wide approaches to protein–protein interaction analysis.

Keywords:
Biochemistry / History / Networks / Protein–protein interactions / Signaling pathways
/ Systems biology

1 Introduction into physical motion, and maintaining cellular organiza-

Proteins play central roles in all aspects of cellular func-
tion including metabolism, information processing, deci-
sion making, transport, and structural organization. Pro-
teins mediate their functions by physically interacting with
proteins and other molecules such as metabolites, lipids,
and nucleic acids. Protein–protein interactions play key
roles for mediating functions such as sensing the environ-
ment, mediating signal transduction, adjusting the activ-
ity of metabolic and signaling enzymes, converting energy
Correspondence: Dr. Pascal Braun, Department of Plant Sys-
tems Biology, Center for Life and Food Sciences Weihenstephan,
Technische Universität München, 85354 Freising, Germany and
Helmholtz Centre Munich, Research Unit Protein Science, In-
golstädter Landstraße 1 85764 München/Oberschleißheim, Ger-
many
E-mail: pbraun@wzw.tum.de
Fax: +49-8161-71-2886
Abbreviations: AP-MS, affinity purification coupled to mass spec-
trometry; HT, high-throughput; IP, immunoprecipitation; MPF,
maturation promoting factor; PKA, protein kinase A; SV40, simian
vacuolating virus 40; Y2H, yeast-two-hybrid
tion. In recent years, it has become apparent that proteins
form intricate networks via their interactions with other pro-
teins, resulting in highly organized, dynamic cellular sys-
tems [1]. Analysis of such interactome networks has provided
important insights into the global organization of cellular
systems.
Because of the critical importance of protein interactions,
when an unknown protein is investigated today, one of the
first questions asked is: what other proteins does it inter-
act with? This was not always the case. The study of protein
interactions started slowly and evolved considerably, hand-in-
hand with conceptual and technological progress in different
areas of biological research (Fig. 1). In this process, no single
point can be identified at which the importance of physical
protein–protein interactions was suddenly realized. Instead,
again and again physical interactions were found to play key
roles in processes that eventually included essentially all bi-
ological functions. We think it is timely to revisit how we
came to recognize the diverse roles exerted by protein–protein
interactions. In this narrative, we present some of the key ex-
periments that have introduced major conceptual changes
Colour Online: See the article online to view Figs. 1 and 4 in colour.


C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
Figure 1. Timeline of protein-protein interaction research. In the top part conceptual advances and discoveries are indicated, in the lower
part technological advances and inventions are indicated.
in biochemistry and molecular biology, and review techno-
logical breakthroughs that have paved the way for today’s
systems-wide approaches to protein interaction analysis. We
apologize for the unavoidable omission of findings due to
space limitations.
2 Proteins, enzymes, and first protein
interactions
The first protein interactions were discovered even before
there was a clear understanding on what proteins are. The
term “protein” (gr. proteios–of the first kind) was proposed
in 1838 by Berzelius in a letter to the Dutch chemist, Mulder.
Mulder had found the same chemical formula for organic
material from plants and animals (C400 H620 N100 O120 P1 S1 ),
which led him and Berzelius to the assumption that this
organic material was made of a single type of molecule [2].
At this point, it was unclear, however, how this substance
related to biological processes. In the same decade, it was
shown that alcoholic fermentation was caused by yeast. Louis
Pasteur later described that different types of fermentation
all require intact cells and considered fermentation a process
irreducibly associated with life [3]. This was often taken as
evidence for the view that living organisms contain a vital
force that was distinct from physicochemical processes (“vi-
talism”) [4]. However, biological activities that acted outside of
organisms had already been identified in the early 1830s. The

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1479
first such activity, diastase, which catalyzes the conversion of
starch to sugar, was discovered in 1833 in an alcohol precip-
itate of malt extract by French chemists working in a sugar
factory [5]. Soon after, a digestive activity in animal stomach
was discovered and named pepsin (1835) [6]. In 1876, Kühne
proposed the term “enzyme” for such ferments that were ac-
tive independent of an organism [6]. In the same article, he
described the isolation of trypsin – the digestive enzyme of the
pancreas [7], which became a member of one of the earliest
protein interaction pairs. In 1906, a trypsin inhibiting activity
was detected in the albumin fraction of blood serum [8]. In a
quantitative kinetic study, the nature of this interaction was
analyzed. Assuming an interaction according to the law of
mass action, i.e. two soluble molecules, the activity of trypsin
was quantitatively predicted for several experiments (Fig. 2).
Importantly, the rate at which the enzyme activity decreased
when either pure enzyme or enzyme-inhibitor complex was
diluted was slower for the latter. This could be explained by
dissociation of the enzyme-inhibitor complex and this result
led to the conclusion that in contrast to inhibition by absorp-
tion to charcoal, the inhibitor and the enzyme “combine to
form an inactive but dissociable compound” [9]. This interac-
tion between the enzyme trypsin and its inhibitor is thus one
of the first regulatory protein–protein interactions identified.
Despite these early insights, it took well into the 20th century
until it was convincingly shown that enzymes are proteins.
In 1926, Sumner purified the major protein in a preparation
of the enzyme urease such that it could be crystallized. Upon
www.proteomics-journal.com
1480 P. Braun and A.-C. Gingras
dissolving the protein crystals in water, the solution exhib-
ited very high enzymatic activity thus demonstrating that the
crystallized protein was identical to the enzyme [10].
Another of the earliest protein–protein interactions was
detected in the context of investigating infectious diseases:
the interaction between antibodies and antigens. In the sec-
ond half of the 19th century, it was known that some infec-
tious diseases are caused by toxins secreted by pathogens. In
the absence of sufficient quantities of pathogen toxins, many
scientists explored the physiological response of animals to
plant toxins. In 1891, Paul Ehrlich published results on the
closely related and highly toxic plant proteins, ricin and abrin
[11]. In the first study, he reported the ability to immunize
mice against the toxic effects of ricin. He explained this by
the formation of an antitoxin, antiricin, in the blood of the
animals. The existence of antiricin was demonstrated by tem-
porarily transferring immunity to naı̈ve animals by injecting
them with antiricin containing blood. In the second article, he
conducted a detailed toxicological study of the closely related
abrin. Both ricin and abrin led to similar toxicologic pheno-
types and had been proposed to be identical. In his studies,
Ehrlich found a number of differences between the two sub-
stances to support the conclusion that they were likely differ-
ent. As with ricin, however, he found that it was possible to
immunize animals against abrin by feeding nontoxic doses,
resulting in the appearance of antiabrin in the blood of the
animals. Crucially, despite extensive pathological, and, as we
know today, extensive structural similarities, animals that are
immune against one toxin are still fully sensitive toward the
other. Ehrlich introduced the term “antikörper” (antibodies)
to define the class of substances with the antitoxic activities
that are produced by the body. Mirroring today’s widespread
use as a technological tool for identification and highly spe-
cific purification, Ehrlich used antibodies as a tool to establish
identity between two proteins “if two substances give rise to two
different antikörper, then they themselves must be different” [12].
It took another 30 years, however, until it was shown that
antibodies are in fact proteins [13].
Shortly after Ehrlich’s discovery of antibodies, in 1897,
Buchner demonstrated that fermentation of sugar can take
place in extracts from yeast lysate [14], i.e. in the absence of

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
Figure 2. Complex formation between
trypsin and trypsin inhibitor. (A) Trypsin
activity is measured by the change in con-
ductivity of a gelatin (substrate) solution
caused by trypsin activity during a de-
fined time interval. (B) Enzyme activity in
different conditions is predicted assuming
a reversible equilibrium between trypsin
and its inhibitor according to the law of
mass action. Calculated and observed en-
zymatic activities for dilutions of trypsin (I)
and trypsin with inhibitor (II) by the indi-
cated volume of water. (Original data from
Northrop, 1922 [9])
living cells. This ultimately disproved vitalism and set the
foundation for modern biochemistry. By the turn of the cen-
tury, enough evidence had accumulated to suggest that en-
zymes could be “the chemical basis of life” [15]. With this,
understanding the molecules and chemical processes of life
became essential for biology. Although physical interactions
among proteins had been identified, this did not immediately
trigger intense research in their functions. First, the molecu-
lar nature of proteins needed to be elucidated which became
a central topic in the new field of biochemistry.
3 The nature of proteins
For a long time, the nature of proteins and their relation-
ship to enzymes was unclear. Due to their molecular weight,
which is orders of magnitude larger than any then known
molecule, many scientists doubted that proteins could be in-
dividual molecules [16–18]. In the 1910s and throughout the
1920s, it was hotly debated whether proteins might be col-
loids: heterogeneous, noncovalent compound assemblies of
smaller molecules and electrolytes [16–18]. Toward the end of
the 1920s, significant progress was made in elucidating the
nature of proteins and their relation to enzymes. One impor-
tant finding was that proteins can be purified and crystallized,
which was the requirement for Sumner’s demonstration that
enzymes are proteins (see above) [10]. The invention of ul-
tracentrifugation by Svedberg contributed decisive evidence
that proteins were in fact large molecules with defined chem-
ical properties. In contrast to the diffuse appearance of even
the most homogeneous colloids, proteins yielded a single ho-
mogeneous border in these experiments (Fig. 3A) suggesting
that “notwithstanding their size and enormous weight, the hemo-
cyanin particles are entitled to be considered as real molecules
acting as single units” [19, 20]. During his centrifugation ex-
periments, Svedberg also noticed that several sharp bands
became visible at higher pH, corresponding to proteins of
smaller molecular weight (Fig. 3B). Upon detailed analysis
of hemocyanins from several species, he realized that the
molecular weights of large species always corresponded to
multiples of the smallest detectable protein, suggesting that
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
A the development of methods to label the proteins after
B but may consist of mixtures of closely related substances with no
Figure 3. Demonstration that hemocyanins are molecules and
consist of subunits. Schematic representation of photographic
plates of ultracentrifuge sedimentation experiments. Sedimen-
tation is performed at constant centrifugal force, picture series
represent 5 min intervals, except bottom left 10 min. (A) Sedi-
mentation of hemocyanins (top) and gold colloids (bottom). Gold
colloids, among the most homogeneous colloids known, sedi-
ment with a diffuse appearance. In contrast, proteins exhibit a
sharp boundary suggesting they are a single type of molecule.
(B) Sedimentation of hemocyanins at lower pH (top) and higher
pH (bottom). Original experiments were done with hemocyanins
from different species in [22]. At lower pH a single sharp sedi-
mentation front is observed. When the pH is increased (bottom)
several sharp fronts corresponding to protein species of lower
molecular weights, later shown to be subunits, become visible.
(Data adapted from Svedberg, 1929 [20]; and Inga et al., 1936 [21])
small subunits aggregate to form larger protein complexes.
Moreover, the apparent dissociation was reversible [19, 21].
Without a conceptual distinction between peptide chains and
proteins at that time, he hypothesized that different proteins
might be assembled from few elementary subunits of approx-
imately 34kDa, which he proposed as a “general plan” about
the nature of proteins [20, 21]. In hindsight, these results are
among the first examples demonstrating that proteins can
exist as dissociable complexes in vivo and mark the discovery
of quaternary structure [22].
If proteins were in fact macromolecules of unprecedented
size, questions about their composition and assembly
became pressing. In the absence of a mechanism by which
highly complex peptide sequences could reproducibly be
synthesized by the cell, protein models in which peptides
are arranged in a repetitive manner seemed reasonable [23].
In the late 1940s, Frederick Sanger was able to determine
the sequence of the insulin B chain. This had required

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1481
partial acid hydrolysis (and in some case cleavage by pro-
teases), fractionation of the products, followed by end-group
identification [24–30]. This was a tedious method, with no au-
tomation, random cleavage of the protein chain, and manual
assembly of protein sequences from small peptide sequences
identified. While the approach could not easily be applied
to larger proteins, the sequencing of insulin unambiguously
demonstrated the noncolloidal, unique, and nonrepetitive
nature of protein primary structure, which Sanger himself
summarized in a 1952 review on this topic: “It has frequently
been suggested that proteins may not be pure chemical entities
absolute unique structure. The chemical results obtained so far
suggest that this is not the case, and that a protein is really a single
chemical substance, each molecule of one protein being identical
to every other molecule of the same pure protein” [31]. In the fol-
lowing year, Sanger and Thompson completed the sequence
of the insulin A chain, which they found to be different from
that of the B chain, leading them to conclude: “It would thus
seem [ . . . ] that each protein has its own unique (amino acid)
arrangement; an arrangement that endows it with its particular
properties and specificities and fits it for the function that it per-
forms in nature.” [32,33]. The chemical nature of proteins was
settled.
Once proteins were shown to consist of amino acids
that are arranged in a specific sequence in each protein, an
important question became how the different amino acids
chains were arranged in three-dimensional structures to yield
molecules that have biological activity. Another monumental
step enabled such structural analysis of pure protein crystals
subjected to x-rays: the conceptual solution of the “phase prob-
lem” by Max Perutz in the early 1950s (see also [34]). Together
with the arrival of computer technology, this enabled solving
the three dimensional structures of myosin and hemoglobin
in 1959 and 1960, respectively [35, 36]. These structures illus-
trated visually how the primary sequence of proteins folds into
a unique three-dimensional shape. Moreover, in the struc-
ture of hemoglobin, the oxygen-carrying protein of red blood
cells, the tetrameric subunit composition of the holoprotein
was apparent, which provided an atomic perspective into the
quaternary arrangements of polypeptide chains in a protein
complex and thus provided an explanation for Svedberg’s
observation of the subunit dissociation of the evolutionary
related hemocyanins. Conceptually, the structures laid the
groundwork for understanding of allosteric interactions [37],
and recognition of specific interaction interfaces [36, 38].
With these landmark papers, together with publication of the
double helix a few years earlier [39], the world of biochemistry
acquired a new dimension of visual accessibility.
4 Interactions give structure and regulate
enzymes
Despite the early identification of interactions between
trypsin and its inhibitor and that of antibodies with their
www.proteomics-journal.com
1482 P. Braun and A.-C. Gingras
antigens, investigation of physical interactions between pro-
teins played a minor role throughout the early 20th century.
Starting in the 1940s, however, more and more examples
of protein–protein interactions were uncovered, and the im-
portance of physical associations between proteins started to
become appreciated.
4.1 Physical motion mediated by interactions
between myosin and actin
Physical motion has intrigued humans since antiquity, when
Aristotle first described how muscles cause motion [40]. In
addition to electrophysiological studies on intact muscle [41],
myosin was identified as a major molecular constituent of
muscles by Kühne in the middle of the 19th century. In the
1930s, studies of myosin revealed its elongated shape [42]
and ATPase activity [43–45]. Importantly, the conformation
of myosin, measured by its viscosity, was shown to reversibly
change upon addition of ATP, which suggested “for the first
time” that myosin was a “contractible enzyme” [46]. In the fol-
lowing years, it became apparent that one of the previously
described forms of myosin, “Myosin B”, was likely a com-
plex of actin and myosin, which physically interact to yield
actomyosin [47–49] (Fig. 4). The physical interaction between
F-actin and myosin was later directly demonstrated in im-
munoprecipitation (IP) experiments [50]. Moreover, it was
shown that the interaction with actin triggers the ATPase
activity of myosin and thus, that the physical interaction be-
tween actin and myosin is critical for enzymatic activity [47].
Thus, “the modern era (of understanding motion) began with
the demonstration that contraction is the result of the interaction
of two proteins, actin and myosin with ATP [ . . . ]” [51].
Actin, the second major protein of muscles, had been iden-
tified in 1887 by its ability to coagulate myosin. However, only
after the realization that Myosin B was a complex of two pro-
teins, actin was purified, named, and characterized in 1942. It
was found to exist in two distinct forms, filamentous (F-) and
globular (G-) actin [51–54]. In 1943, it was demonstrated that
G-actin could be obtained by desalting and thus depolymer-
izing F-actin [53]. The immunologic identity between both
forms was further supported by the mutual recognition of
each actin species by antibodies targeting the other form
[50]. In 1950, Straub demonstrated that removal of ATP from
G-actin prevented polymerization into F-actin [54–56], and
thereby, smooth muscle actin polymerization became an early
example of an ATP-driven protein–protein interaction. Over
the next years, additional interactors for actin and myosin
were identified, and the mechanism by which physically in-
teracting proteins mediate physical motion was elucidated
[51, 57, 58]. Conceptually, actin fibers were also the first ex-
ample for cytoskeletal structures. In the following decades,
the ubiquitous role of physical protein–protein interactions
in creating diverse subcellular structures became apparent
following the discoveries of microtubules and intermediate
filaments, as well as the nuclear pore complex. These struc-

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
tures mediate a wide range of biological functions related to
both structural organization and motility including cellular
motion, intracellular cargo transport, and exertion and sens-
ing of mechanical forces and are a topic of intense research
to this day [59].
4.2 Protein interactions regulate metabolism
In the mid 1940s, the understanding of metabolism was rev-
olutionized by conceptual and technical advances. In 1944,
Beadle and Tatum published the one-gene-one-enzyme hy-
pothesis, and demonstrated how genetic screens can be used
to identify new enzymatic reactions by isolation of mutants
that lost the ability to grow on media lacking specific metabo-
lites [60]. Although initially met with resistance and deemed
irrelevant by some of the most eminent geneticists at the time,
their results demonstrated that complex processes such as
metabolism were amenable to genetic analysis [61]. In subse-
quent years, genes encoding enzymes in numerous biochem-
ical pathways were identified. At the same time, an increasing
number of metabolic enzymes was purified and investigated
in biochemical detail. For many of these, molecular weights
in the megadalton range were observed, which was very large
even for proteins. For example, purified ␣-ketoglutaric oxi-
dase was found to sediment with a molecular weight of two
megadaltons, suggesting that “[ . . . ] large molecules of pyruvic
and ␣-ketoglutaric oxidase may still be complexes of more than one
protein. If so, they would represent organized units below the mito-
chondrial level” [62]. Despite this early and correct speculation,
it took nearly another 10 years for this enzyme complex to be
resolved into its constituent protein components. Once the
individual proteins were identified, each could be associated
with individual reaction steps of the overall catalyzed reac-
tion [63]. Notably, in reassociation experiments described in
the same paper, the original protein complex could be recon-
stituted from the individual proteins, clearly demonstrating
the noncovalent nature of the interactions. Studies on other
enzymes came to similar conclusions [64]. Over the next 10
years, facilitated by progress in gel electrophoresis, the com-
position of more and more enzymes was determined. In light
of “hundreds of reports” describing the molecular weights of
enzymes and their subunits, in 1971 it was noted that “it
would appear that the vast majority of proteins are composed of
subunits” [65].
The widespread identification of multimeric enzymes gen-
erated an interest in the functional aspects of these subunit
interactions, and evidence for subunit regulation accumu-
lated. In 1965, Monod and colleagues developed a model of
allosteric enzyme regulation, i.e. functional interactions of
different ligands that bind to spatially separated, i.e. non-
overlapping binding sites. A central element of the model is
symmetry of different binding sites for a given ligand as of-
ten found in protein complexes. At least two conformational
states of the oligomeric protein complex are postulated; tran-
sition from one conformational state into the other results
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
in a changed affinity of all symmetric binding sites for their
corresponding ligand. With this model, several regulatory
phenomena could be explained and enzymes could be catego-
rized as “K-systems” where an effector F modifies the affinity
of the enzyme for its substrate and “V-systems” where ef-
fector binding has no impact on affinity but alters catalytic
activity. The model quantitatively explained several observed
effects, including the identical allosteric coefficients of carbon
monoxide and oxygen, despite 250-fold higher absolute affin-
ity of the former for hemoglobin [37]. The authors conclude,
that “oligomers (are well suited) as mediators of chemical sig-
nals [ . . . ] (because) certain desirable physical and physiological
properties are associated with symmetry, and therefore inaccessible
to a monomeric protein” [37]. Protein interactions thus were
recognized as playing a central role in the dynamic adapta-
tion of enzyme activity according to metabolic requirements
of the organism [37]. In addition, heterologous interactions
of enzymes with nonenzymatic proteins were identified that
modify catalytic parameters, as demonstrated by the dras-
tic increase in Aldolase activity elicited by interaction with
F-actin [66].
Progress in understanding the importance of protein–
protein interactions led to the idea that perturbation of physi-
cal interactions may underlie genetic complementation, in
particular interallelic complementation [67]. In interallelic
complementation different non- or semi-functional alleles

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1483
Figure 4. Experimental demonstration that
interaction between myosin and actin un-
derlies physical motion. (A) Ostwald vis-
cosimeter used to determine the viscosity
of myosin solutions. Liquid is drawn to the
upper mark and released. From the time
required for the liquid level to fall to the
lower mark the viscosity of the liquid can
be calculated. (B) From ground muscle tis-
sue myosin is extracted using high salt so-
lution. Extraction for 20 minutes results in
myosin A, which is characterized by low vis-
cosity, whereas extraction over night results
in myosin B with high viscosity. Addition of
ATP results in decrease of the viscosity of
Myosin B. In protein purifications myosin
B was later shown to consist of actin and
myosin. (C) Myosin solution squirted into
water leads to formation of myosin fibers.
Drying results in myosin fibers structurally
similar to structures seen in electron mi-
crographs of intact muscle [52]. (D) Fibers
are prepared from myosin A and B prepara-
tions. Addition of ATP to actomyosin fibers
results in in vitro contraction. (Illustrations
based on Szent-Gyorgyi, 2004 [51]).
of the same “cistron”, i.e. same gene, can complement and
hence rescue each other. Several models to explain this phe-
nomenon were proposed. The simple model in which com-
plementing mutations at the protein interaction interface en-
able an otherwise not feasible interaction, was deemed un-
likely based on the frequency of observed complementation
patterns [67]. Instead, it was proposed that correction acts dis-
tantly, i.e. not at the interface, but, by mutual correction of the
overall folding of the involved subunits [67]. A general version
of this argument was developed by McGavin in 1968 based on
the allosteric model by Monod and colleagues [68]. McGavin
considered a multimeric symmetrical protein complex pre-
disposed to assume different conformational states. In a ho-
momeric complex of subunits encoded by a single allele, the
“damaged” sites are necessarily symmetrical and hence rein-
force each other in stabilizing one or the other conformation.
In contrast, in a heteromeric complex composed of subunits
encoded by different alleles, mutated sites are unsymmetrical
and hence may balance each others effects [68]. McGavin con-
cluded that intraallelic complementation should arise with a
given frequency, which would also imply evolutionary advan-
tages of organizing proteins in multimeric complexes [37,68].
To this day, the understanding of enzyme regulation by
protein–protein and protein–metabolite interactions is a topic
of great interest. Moreover, the recent abundance of system-
atic genetic data on human disease and plant traits relevant
www.proteomics-journal.com
1484 P. Braun and A.-C. Gingras
for food production, have increased the urgency for under-
standing how genetic variation is mediated on the protein
level to give rise to pathologic or desirable traits (see below).
4.3 The first signal transduction cascade
During the 1960s, at a time when phosphoproteins were con-
sidered “biologically inert and ( . . . ) uninteresting” [69], the
study of hormone-induced glycogen release led to the discov-
ery of the first signal transduction cascade. Work in the 1940s
in Carl and Gerty Coris’ lab had identified glycogen phospho-
rylase (hereafter phosphorylase), the rate limiting and highly
controlled enzyme that catalyzes the first step in the conver-
sion of glycogen to glucose. They found two interconvertible
versions of this enzyme; phosphorylase b, which requires ac-
tivation by adenosine monophosphate (AMP) and phospho-
rylase a, which they assumed had AMP covalently bound as
a prosthetic group. While Coris’ had observed the conversion
of phosphorylase a to b [70], and even identified an activity cat-
alyzing it, their mechanistic explanation of AMP being a pros-
thetic group, which was removed by the prosthetic group re-
leasing (PR) enzyme [71], was inconsistent with the absence of
nucleosides in crystallized phosphorylase b preparations [69].
Fisher and Krebs turned to study phosphorylase in the 1950s
and realized that activation required ATP instead of AMP,
which led them to discover that the activated phosphorylase a
contained isotopic phosphate when incubated in the presence
of ␥-32 P-ATP. Phosphorylase thus became the first example
of an enzyme regulated by phosphorylation. Structural and
functional analysis later revealed that phosphorylase a is pre-
dominantly a tetramer, whereas the b form occurs mostly as a
dimer [72]. Thus, phosphorylase must also be considered the
first example of a phosphorylation-regulated protein–protein
interaction. Development of an in vitro assay [73] enabled
purification of the upstream kinase (phosphorylase kinase);
the previously described phosphorylase a to b converting en-
zyme was identified as the corresponding phosphatase (pro-
tein phosphatase 1 or PP1) [74–76]. This early work was fol-
lowed years later by crystallization studies, paving the way
for the study of phosphorylation-regulated protein–protein
interactions (see below). Physiologically, however, important
questions remained. Sutherland had shown that hormone
stimulation of cells is followed by the production of cyclic
AMP (cAMP) [77, 78], which was critical for the activation
of phosphorylase [77]. While phosphorylase kinase directly
regulated phosphorylase, it did not itself bind cAMP, and
therefore could not be its sensor. This motivated the search
for a cAMP-dependent kinase activity, which was identified
by the Krebs laboratory in 1968 [79]. The purified protein
became known as PKA (protein kinase A). As already spec-
ulated in the original publication, PKA could phosphorylate
and thereby activate phosphorylase kinase [80]. Thus PKA
provided a key link between the hormone-induced produc-
tion of cAMP and the activation of metabolic processes; at

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
the same time the discovery of PKA established the first sig-
nal transduction cascade.
After its discovery, PKA became an intensely studied
protein. In the early 1970s, it was shown that PKA consists
of a tetramer of two catalytic subunits associated to two reg-
ulatory subunits that act as the cAMP sensors [81–83]. Thus,
physical interactions were shown to play a critical role in
regulating the activity of signaling proteins. Around the same
time, proteins controlling the specificity of phosphorylase
phosphatase (PP1) via physical interactions were identified;
studies from Cohen and colleagues identified both protein
phosphatase inhibitors as well as targeting subunits for PP1,
including a targeting subunit for glycogen and a different
one for myosin [84].
A critical element of signal transduction cascades that re-
mained missing for a long time, were the hormone- and
growth factor receptors, including those that activated adeny-
late cyclase and PKA. Fascinatingly, even though receptors
had been studied pharmacologically since the 1920s (see
[85,86]) and detailed theoretical models had been developed to
quantitatively describe ligand binding [86], up until the 1970s
the physical existence of receptors was controversial [85, 87].
A critical step forward was the development of radiolabel-
ing methods during the 1960s: Isotopic iodine was covalently
attached to receptor agonists and thus enabled quantitative
determination of ligand binding constants and of receptor
molecules on the cell surface. These experiments progressed
in parallel for growth factor receptors [88, 89], ion-channel
receptors [90], and G-protein coupled receptors [91]. Radi-
olabeled ligands also enabled the purification of receptors,
by tracing their presence during biochemical purification.
Using such an approach, the nicotinic acetylcholine recep-
tor, which is especially abundant in the electric organs of
electric ray (Torpedo marmorata) and eel (Electrophorus elec-
tricus), was first purified [92]. Injection of rabbits with puri-
fied receptor resulted in production of antibodies followed by
paralysis of the animals [93, 94]. This result was critical for
establishing the functional relevance of the purified agonist-
binding proteins and establishing that receptors and ligands
were proteins that physically interacted. SDS-PAGE analysis
of purified receptors further revealed that they were com-
posed of five subunits [95], a result that laid the groundwork
for understanding the structural organization and allosteric
regulation of ion channels [87]. In the following years, an in-
tense search and rapid identification of other kinases, phos-
phatases, and other downstream effectors of receptors such as
heterotromeric G-proteins [96] ensued in many different dis-
ciplines. Signal transduction research was born, and protein–
protein interactions took a more central role.
5 Interactions in time and space
After the fundamental concepts of molecular biology had
been laid out, technical innovations in many fields in the
late 1960s and early 70s revolutionized the analysis of
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
molecular biological events. The optimization of cell culture
by virologists [97], development of SDS gel electrophoresis
by biochemists [98], development of monoclonal antibody
technology by immunologists [99–104], refinement of genetic
screens, and the development of molecular cloning by molec-
ular biologists [105] provided researchers with a toolbox of
unprecedented capability. Empowered by genetics but chal-
lenged by the enormous complexity of human disease, most
notably cancer, simpler systems were adopted for the study
of the eukaryotic cell cycle and the molecular causes of cancer
induced by oncogenic viruses. These studies led to major ad-
vances in the conceptual appreciation of the central role and
omnipresence of regulated protein interactions.
5.1 Protein interaction controls the eukaryotic cell
cycle
Cell division was recognized as one of the most defining char-
acteristics of life ever since Virchow concluded that living cells
can only arise from other cells [106]. At the turn of the cen-
tury, embryologists and cytologists had described the macro-
molecular events of cell division using light microscopy, but
investigation into the molecular underpinnings of this pro-
cess started to bear fruit in the 1970s. Exploration of cell cycle
control was approached in two completely different systems
in initially separated communities. Geneticists used muta-
tion analysis in yeast to identify temperature-sensitive mu-
tants that arrested the cell cycle at specific points [107, 108].
Hartwell and colleagues identified a series of cell division
control (CDC) mutants, amid them the budding yeast CDC28
gene and its fission yeast homologue cdc2. Complementation
screening of cdc2 mutants in Paul Nurse’s lab enabled iso-
lation of the coding sequence for cdc2, which was found to
encode a protein kinase [109, 110].
In parallel, biochemists investigated the cell-cycle in
oocyte extracts. They observed the breakdown of the nuclear
envelope, a marker for cell division, when activated cytosolic
extracts were added to those from inactive oocytes [111]. A
critical biochemical activity, named maturation promoting
factor (MPF), was identified as key factor for this process
[112]. Tim Hunt’s lab later described a protein that appeared
and disappeared in phase with the cell cycle, and which was
therefore named cyclin [113]. Even though the first cyclin
gene was cloned shortly after isolation of the cdc2 cDNA
[114], by the mid 1980s, the cell cycle studies in oocyte
extracts and yeast stood disconnected next to each other, as
did cyclin and kinase.
At that time, the widespread of homology between proteins
of evolutionary distant organisms was not yet recognized and
it was assumed that the mechanisms regulating the cell cy-
cle in uni and multicellular organisms were fundamentally
different [115]. This view was shattered when experiments
in Paul Nurse’s lab identified a human cDNA that comple-
mented the yeast cdc2 mutant. The isolated cDNA not only
encoded a kinase that exhibited 63% homology to the yeast

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1485
cdc2p protein; antibodies raised against the yeast cdc2p pro-
tein were able to recognize a single band in human cells
[116]. This strongly suggested that closely related proteins
were involved in regulating the cell cycle in evolutionary
distant organisms [115]. However, the connection of these
kinases to MPF was still unclear. By the end of the 1980s, it
became possible to purify MPF, which was found to consist of
two proteins. Fascinatingly, the very same antibodies against
yeast cdc2p recognized the frog orthologue of cdc2p as the
first component of MPF [117, 118]. The second protein was
found to be the oscillating cyclin A [119], which activated the
kinase in a cell-cycle dependent manner by physically inter-
acting with it [120]. Thus, by the end of the decade, orthogonal
approaches in very different model systems had resulted in
elucidation of the basic concepts of the universal eukaryotic
cell cycle, at the core of which stood the interacting protein
pair of a cyclin and a cyclin-dependent kinase (cdc2) [121].
5.2 Protein interactions’ role in transformation
by oncogenic viruses
In parallel to the cell-cycle investigations, the molecular basis
of cellular transformation by oncogenic viruses was intensely
studied and led to several important insights about the roles
and regulation of protein interactions. By the early 1970s,
geneticists had identified viral genes required for transfor-
mation by Rous sarcoma virus (RSV), adenovirus, Simian
vacuolating virus 40 (SV40), and others. For SV40, a tumor-
specific antigen (large-T) had been discovered in 1963, and
by the late 1970, several associated proteins were known
[64, 122, 123]. The significance of these interactions for cellu-
lar transformation was unclear, though. For RSV, attempts to
raise antibodies against a tumor antigen succeeded in 1976,
and enabled isolation and characterization of the oncogenic
v-src protein kinase [124–126]. Identification of a homolo-
gous kinase encoded in the host genome (c-src) [126], and
recognition that the viral protein was a constitutively active
version of the proto-oncogenic host kinase, later suggested
that similar molecular mechanisms underlie etiologically dif-
ferent cancers [126]. In 1979 a kinase activity, later shown
to be c-src, was detected in IPs of the SV40 large-T antigen
[127]. Surprisingly, this kinase preferentially phosphorylated
tyrosine residues. Until then, only phosphorylation on serine
and threonine was documented in eukaryotes. It was there-
fore unclear whether the identified phosphotyrosine, which
is recognized today as one of the most significant posttransla-
tional modifications in growth factor signaling, was the end-
product of the reaction or “merely an intermediate of a reaction
that could not run to completion in the IP” [127]. Shortly af-
ter, however, tyrosine phosphorylation by epidermal growth
factor receptor (EGFR) and v-Abl was described [128, 129],
and the initial caution gave way to an intense search for tyro-
sine phosphorylated substrates. With the growing availability
of sequence information, computational approaches such as
sequence alignment became increasingly useful [130, 131].
www.proteomics-journal.com
1486 P. Braun and A.-C. Gingras
Using a combination of experimental and computational
analysis, a conserved noncatalytic domain critical for trans-
formation by src was identified by the Pawson group in 1986
[132]. While experimental data indicated the importance of
the identified domain for transformation, its molecular func-
tion was unclear. However, in 1990 the same group demon-
strated that homologous domains (src homology 2, or SH2
domains) from several important signaling proteins could
bind to activated, but not to unstimulated, EGFR [133]. Spec-
ulation that the interactions might be regulated by tyrosine
phosphorylation was later confirmed [134]. This discovery not
only demonstrated that dedicated domains mediate posttrans-
lational modification-dependent interactions, but also sug-
gested that dynamic regulation of protein interactions was a
more common phenomenon than was appreciated until then.
In subsequent years, numerous other modification-specific
interactions involving structurally diverse protein domains
were identified [135], and the immense combinatorial poten-
tial for signal processing, achieved by concatenating different
interaction domains, was recognized.
By the late 1980s, work on the src oncogenes had iden-
tified growth promoting host pathways and suggested that
retroviruses cause cellular transformation by activating these
pathways. Despite the presence of src activity in SV40 large-T
IPs though, the mechanism by which DNA tumor viruses
such as SV40 and adenovirus caused transformation re-
mained largely elusive. Several physical interaction partners
for adenovirus E1A and large-T antigen, identified as bands
on SDS-PAGE gels of IPs had been characterized and addi-
tional proteins remained to be studied. One of the unknown
proteins found in IP of the adenoviruses E1A oncoprotein
had a molecular weight of approximately 105 kD. Around the
same time, the Weinberg laboratory had identified the first
example of a new class of cancer genes–the retinoblastoma
tumor suppressor gene (Rb). The molecular function of the
encoded 105 kD retinoblastoma protein (pRb), however, was
unclear. Reciprocal IPs using antibodies against pRb or the
E1A-associated protein revealed the identity of both proteins,
resulting in the first characterization of a physical interaction
between a viral oncogene and a human tumor suppressor
gene in 1988 [136]. This suggested that both positive and neg-
ative growth regulators do not necessarily function through
separate pathways but “may directly confront one another
through the interaction of their encoded proteins” [136]. In subse-
quent years, additional examples of this “direct confrontation”
were discovered, including interactions of viral oncogenes
with pRb and p53, which in 1989 was recognized as a tumor
suppressor protein [137], and interactions between pro and
antiapoptotic members of the BCL-2 protein family [138].
5.3 Spatial control of interactions in signaling
modules
In addition to the temporal regulation of signaling, the im-
portance of spatial regulation became apparent during the
continued investigation of PKA. By the 1980s, PKA activation

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
had been observed following a wide array of stimuli [139,140].
At a time when the cell was still considered an unstructured
“bag of enzymes” [141] the fact that a single kinase can mediate
a diversity of signals, some of these causing opposite phys-
iological effects, was puzzling [141]. More detailed analysis
revealed that different external signals can selectively activate
a subpopulation of PKA molecules [142]. Subsequent anal-
ysis of the underlying molecular mechanism revealed that
type II-B PKA was tightly bound to microtubules via a phys-
ical interaction with a microtubule-associated protein [143].
Genetic experiments confirmed that the physical interactions
with such A-kinase anchoring proteins (AKAPs) are critical
for the observed selective activation and the resulting physio-
logical specificity of PKA signals [144]. AKAP-PKA modules
were the first example of signaling modules that are spatially
insulated from each other by physical protein–protein interac-
tions [139]. The concept of compartmentalization has become
a major topic in PKA research, and soon encompassed other
kinases and signaling molecules, including raf-ras-MAPK sig-
naling [145]. This remains an active area of research and more
compartmentalized signaling structures are likely to be iden-
tified in coming years [141].
Thus, in the 1970s and 1980s, a multitude of technical
advances and the confluence of the hitherto largely sepa-
rated fields of genetics, biochemistry, and cell biology brought
about a dramatic boost in the understanding of molecular cel-
lular processes. In the course of these experiments, physical
protein interactions appeared to play key roles in all investi-
gated processes, including the molecular events underlying
one of the most deadly human diseases. These advances in
the conceptual understanding of the importance of protein
interactions were brought about by numerous technological
innovations that facilitated interaction analysis and later acted
synergistically with the completion of genome sequences for
several organisms.
6 Genome ready technologies
At the beginning of the 1990s, protein interactions were
widely recognized as critical for most cellular processes, and
additional technological innovations facilitated their analysis.
This resulted in an explosion of papers describing interacting
protein pairs (Fig. 5). An important innovation, which set the
foundation for essentially all currently used high-throughput
(HT) interaction assays, was the application of gene fusions
to the study of proteins. The first gene fusions had been used
to study gene regulation [146]. In the 1980s, the technology
was applied to proteins: a cDNA for the protein of interest was
genetically attached to a second cDNA encoding a protein that
provides additional functionality. The “hybrid protein” solved
a nagging problem at the time when genes involved in diverse
processes were being identified, but no biochemical assay or
antibody existed for the encoded proteins, which largely pre-
vented their purification and biochemical characterization. In
1979, the first hybrid protein was described, consisting of a
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
Figure 5. Number of publications published each year on the
topic "protein-protein interaction". (Source: Science citation in-
dex)
bacterial protein of unknown function and β-Galactosidase,
which could be used to detect and purify the hybrid protein
using a routine colorimetric assay [147]. The genetic fusion
with a partner exhibiting high affinity for a ligand provided the
additional advantage that an affinity step could be included in
the purification procedure [148]. The power of this approach
was quickly realized, and many dedicated affinity-fusion tags
were developed, including glutathione-S-transferase (GST),
which simplified elution with an economical, dialyzable elu-
ent [149]. Combination of fusion tags, were also introduced,
which enabled the generation of proteins of higher purity
and led to the development of tandem affinity purification
for proteome-scale protein complex analysis [150–153]. Fu-
sion proteins with peptide sequences recognized by specific
monoclonal antibodies, e.g. the Hemaglutinin (HA) peptide
of the influenza virus (HA-tag) [154], enabled both detection
and purification of the protein using immunological proce-
dures, and hence greatly facilitated the characterization of
proteins and the identification of their interactions.
The value of protein purification for discovery of new in-
teraction partners rests in the ability to identify the unknown
partner. This can be done using techniques such as Edman
degradation [155] and more recently mass spectrometry (MS).
A limitation of early mass spectrometers was that large macro-
molecules such as peptides or proteins are difficult to ionize.
Toward the end of 1980s, two new ionization methods for
biological samples were introduced, which transformed the
field of biological MS and which are still in use today. Matrix-
assisted laser desorption ionization (MALDI) is based on ion-
ization of molecules embedded in a crystallized matrix by UV
light laser [156]. In electrospray ionization (ESI), a high volt-
age is applied to an analyte containing solvent exiting through

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1487
the tip of a fine capillary directly into the mass spectrometer
[157]. ESI affords several advantages over MALDI, not least
the ability to directly couple the separation of peptide mixture
by high-performance liquid chromatography to their mass
spectrometric identification. The invention of tandem MS en-
abled fragmentation and hence sequencing of single peptides
[158, 159]. However, largely due to the difficulties in de novo
protein sequencing by MS, Edman degradation remained a
critical technique for the de novo identification of unknown
proteins until the beginning of the genomic revolution.
The identification of novel interacting proteins thus was
still challenging. To complement approaches based on the
biochemical enrichment of proteins, a number of alternative
approaches were developed. In 1989, Fields and Song pub-
lished a method that circumvented biochemistry altogether,
and thus greatly facilitated the identification of novel protein
interaction partners: the yeast-2-hybrid (Y2H) system [160].
Building on studies that demonstrated that the DNA bind-
ing and activating functions of the yeast transcription factor
GAL4 can be separated, they developed a system in which a
physical interaction of two proteins was detected genetically
by activation of a selectable reporter in yeast. This straight-
forward system could be used to screen large cDNA libraries
using a simple growth-selection based assay, and novel inter-
actors could be identified using routine DNA sequencing. In
subsequent years, many key interactions were identified by
Y2H, including one between the mammalian oncogene ras
and its downstream kinase raf [161]. A foreshadowing of its
future importance came in 1996, when the Y2H system was
used to construct the first proteome-scale “protein linkage
map”: that of bacteriophage lambda containing 25 interac-
tions [162]. Importantly, the development of Y2H was also
a proof of concept, which encouraged the development of a
large number of technologies for protein interaction analysis
that are based on the reconstitution of a functional protein by
protein–protein interactions [163–167]. Several of these are
described in chapters by Lam and Stagljar, Braun, and Taylor
and Wrana in this issue.
During the 1990s, methods based on affinity purification
of interacting proteins, on genetic approaches such as the
Y2H system and other interaction detection strategies were
employed by many different labs to address questions in es-
sentially all fields of molecular biological inquiry. The true
power of all these approaches, however, needed additional
ingredients to blossom into the power-house technologies
that they are today.
7 The genomic revolution–protein
interaction networks
Against the backdrop of heavy criticism for being too ex-
pensive, “bad big biology”, “technically impossible” and “of
limited scientific value” [168], the Human Genome Project
(HGP) was officially launched in 1990. The resulting ge-
nomics revolution also majorly affected the analysis of
www.proteomics-journal.com
1488 P. Braun and A.-C. Gingras
protein–protein interactions. A key development was the
identification of (nearly) all genes encoded in a genome.
Genomic and expressed sequence tag (EST) sequences pro-
vided a template for the construction of genome-scale cDNA
collections (e.g. the mammalian gene collection, MGC) and
open reading frame (ORF) collections [169–176]. In contrast
to cDNA libraries, in ORF collections only the protein cod-
ing sequence of a transcript is encoded by a plasmid and
not the 5 and 3 untranslated regions of the cDNA. There-
fore, the encoded proteins can be directly fused to tags and
hence can be used in a variety of functional experiments. In
addition, during construction of indexed cDNA or ORF col-
lections, biases and differential representation of clones are
largely eliminated, such that a given sequence is present only
once in the collection. This elimination of redundancy has en-
abled complex experiments, including the analysis of protein
interactions, which would otherwise have been prohibitively
expensive. The development of recombinational cloning fur-
ther facilitated the HT transfer of ORFs into expression vec-
tors for functional experiments, including global Y2H screens
[169, 177, 178]. For MS, the availability of complete gene cat-
alogues provided an additional massive advantage: instead
of sequencing proteins de novo using peptide fragmentation
spectra, which to this day is not automated, the fragmentation
spectra could be matched against the translation products of
all genes using easily automatable algorithms [159].
Interactome analysis thus benefited immensely from the
HGP. At the same time, protein interactions offer an excit-
ing and efficient route to assign biological function to the
thousands of unknown proteins that were uncovered in ev-
ery sequenced genome [179, 180]. With this, the focus of in-
quiry shifted from “which proteins are involved in my biolog-
ical phenomenon?” to “which processes are all the encoded
proteins involved in?” [69], and large-scale protein interac-
tion analysis became an important tool for answering this
question.
8 Interactome networks
Confronted with an immense number of genes encoding
proteins of unknown function, and equipped with automat-
able protein interaction assays, global interaction mapping
experiments started while sequencing of the human genome
was still underway. The first systematic protein–protein in-
teraction maps were published in 2000 for Y2H [181–184]
and in 2002 for AP-MS analysis [185–188], followed soon by
large-scale interaction maps using different technologies for
C. elegans, D. melanogaster, H. sapiens, several microorgan-
isms, and recently higher plants [189–198]. Especially for S.
cerevisiae, which served as test case for essentially all large-
scale approaches, other functional data were collected in a
systematic fashion including localization [199], transcription
[200], phenotypes [201, 202], genetic interactions [203], and
kinase-substrate interactions [204, 205]. These analyses have
led to the realization that other biological properties, e.g. the

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
regulation of mRNA expression or presence in genomes, are
correlated for physically interacting proteins [206]. Many of
the discovered correlations, as well as structural information,
have later been used to predict physical interactions of pro-
teins using bioinformatic approaches [207–209].
The exclusive focus of the first reports describing large-
scale approaches to protein–protein interaction analysis was
development of hypotheses for unknown proteins [180]. Soon
after publication of the first data sets, however, the network
structure and global interconnectivity of all proteins was no-
ticed and the network itself was investigated [210]. Analysis
of protein–protein interaction networks using computational
and statistical tools has since then contributed important in-
sights about the large-scale organization of biological systems
and how networks mediate genotype to phenotype relation-
ships (see also [211]). Structurally, binary interactome maps,
i.e. maps based on binary assays like Y2H that detect pre-
dominantly direct interactions, were found to have a so-called
scale-free topology [212]. In networks of this topology, most
proteins (nodes) have one or few interactions (edges) and few
proteins, so called hubs, have a very large number of inter-
action partners. Networks with this topology, which is also
found for other complex networks such as the internet or so-
cial networks, are resilient against failure of random nodes,
e.g. by mutation, but sensitive to targeted attack of the hubs
[213]. Fascinatingly, in both plants and human, proteins of
viral, bacterial, and fungal pathogens were all found to target
such hub proteins [214–218]. In addition to providing insights
into the structural organization of interactome networks, the
relationship between physical and genetic interactions in bi-
ological systems is becoming increasingly clear. In yeast, a
positive correlation between the number of direct physical
interaction partners of a protein and the number of pheno-
types of the corresponding genetic null was observed [219],
suggesting that at least hubs in binary networks participate
in diverse processes. For human diseases, proteins involved
in a common disease were found more to likely interact than
expected by chance [220]. Conversely, for several diseases, the
protein products of disease causing genes were found to phys-
ically interact and form highly connected disease “modules”
[221, 222]. Further, network analysis has been successfully
used in defining novel cancer and disease genes [223, 224].
Whereas Y2H-based interactome maps revealed the scale-free
topology of the network of direct interactions, AP-MS-based
interactome analysis illuminated the modularity of the in-
teractome and revealed molecular machines [187, 188, 225].
Combining protein complex information with data on ge-
netic interactions in yeast revealed that proteins within com-
plexes are characterized by similar patterns of genetic interac-
tions [226–230]. In recent years, protein–protein interaction
network information has played an increasing role in the
analysis of genetic disease data, e.g. from genome-wide asso-
ciation studies [231, 232]. Moreover, computational integra-
tion of interaction information with transcriptional informa-
tion from tumors was shown to have predictive value for the
classification of tumor samples ([233] and Taylor et al., this
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
issue) Thus, interactome network analysis has become an
important tool for understanding the organization of cellular
systems [211, 234, 235].
In addition to their biological interpretation, data quality
became an important topic in the field of protein interaction
analysis. While initially it was argued that interaction data
from high-throughput approaches are inferior to those ob-
tained in small-scale studies, today it has been shown that
high-throughput approaches can provide data of a quality
identical to those supported by multiple small-scale studies
collected in manually curated public databases [193,219,236].
This is partly due to considerable progress in the concep-
tual understanding of the data produced by different tech-
nologies, and aided by the development of quantitative ex-
perimental approaches to evaluate interactome data sets. For
binary assays, the use of standardized reference sets by dif-
ferent laboratories to benchmark the assays has started to
increase transparency and comparability of data [236–238].
Moreover, development of a quantitative framework for the
evaluation of interactome maps, also based on reference sets,
led to the insight that currently available systematic binary
network maps only cover a small fraction of the respec-
tive interactomes of an organism [193, 219, 236, 237, 239].
By taking into account the limitations of interactome map-
ping experiments, namely the completeness of the ORF col-
lection, the assay sensitivity, and the degree of saturation of
the screen, it is possible to determine what fraction of a
given interactome has been mapped. By this method, the
binary interactomes of yeast, human, and plant have been
estimated to consist of approximately 18 000, 160 000, and
320 000 protein–protein interactions, respectively, of which
between 2% and 20% have been mapped by systematic high-
quality maps [193, 219, 236]. Alternative approaches to es-
timate the interactome size obtain slightly higher, although
overall similar estimates [240,241]. Thus, increasing the over-
all sensitivity of interactome maps without sacrificing data
quality is a pressing challenge in the field as discussed
in more detail by Braun in this issue [167]. For S. cere-
visiae, Y2H-based binary interactome maps were comple-
mented by systematic data sets acquired with other tech-
nologies including split-ubiquitin and protein complemen-
tation assay [196, 197]. The development of standardized in-
teraction assay benchmarking and strategies for systematic
confidence scoring of individual interactions are important
developments that facilitate the comparison and integration
of interactome maps acquired with different technologies
[167, 237].
Interactome analysis using AP-MS is dealing with simi-
lar challenges regarding the need to increase assay sensitiv-
ity, to standardize and compare results produced in different
laboratories, and develop methods for robust and compara-
ble determination of interaction data quality. Single-tag pu-
rifications are increasingly used to capture more transient
interactions and the detection sensitivity of instruments is
improving spectacularly. While this is undoubtedly exciting,
widely accepted and standardized methods to assess data

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1489
quality are critical. Due to the nature of MS data, very dif-
ferent solutions from those for binary assays are being devel-
oped. Quantitative information regarding the abundance of
each prey is embedded in every MS experiment. The use of
this quantitative (label-free) information can be harnessed to
provide confidence score for each bait-prey interaction pair,
by performing modeling either against a set of well-defined
negative control experiments, or across large sets of AP-MS
samples [225, 242–246]. This type of approach is pursued by
several labs and covered in greater detail in the reviews by
Nesvizhskii, and Dunham et al., in this issue. Alternatively,
approaches exploiting isotopic labeling as a means to iden-
tify true interactors have also led to significant breakthroughs
(e.g. [247–250], and reviewed by Trinkle-Mulcahy et al., this
issue).
While most HT AP-MS efforts have thus far focused on the
use of epitope tags as affinity handles, an alternative approach
is to employ antibodies specific for the endogenous proteins
themselves. While a number of difficulties (e.g. procurement
of antibodies to each target and design of appropriate con-
trols) are associated with this approach (reviewed in Dunham
et al., this issue), the growing availability of both natural and
synthetic antibodies as affinity reagents [251–254] enables
researchers to analyze protein interactions in more physio-
logical conditions. Recently, a study using greater than 1800
antibodies against transcription coregulator proteins demon-
strated that this is a viable approach for high-throughput
interaction mapping, provided that stringent data filtering
is applied [255].
9 Protein–protein interactions
and therapeutics
Because of the ubiquity of protein–protein interactions and
the diversity of mediated biological functions, they consti-
tute attractive targets for the modulation of biological sys-
tems. Therapeutic antibodies against secreted proteins and
receptors are an important and rapidly growing segment of
the prescription drug market [256, 257]. Moreover, intracel-
lular modulators of protein–protein interactions, e.g. tubulin
binders such as colchicine or taxol, have been used clini-
cally for many years [258,259]. Nonetheless, developing small
molecule modulators of intracellular regulatory interactions
is challenging [260]. In contrast to the deep substrate bind-
ing pockets of enzymes, interaction surfaces are usually more
flat and extended. Nonetheless, important progress and proof
of concept experiments have demonstrated the general fea-
sibility of targeting protein–protein interactions with small
molecules. The general goal is to identify so-called hot spots
on the surface that either contribute a large portion of the total
binding energy, or are amenable to steric interference with a
small molecule compound [261]. While identification of such
hot spots is often done by scanning mutagenesis, genetic
approaches such as the reverse Y2H, in which interaction-
deficient full-length alleles can be isolated by selection in
www.proteomics-journal.com
1490 P. Braun and A.-C. Gingras
yeast, could provide a cost-effective alternative [262,263]. Even
after identification of hot spots, finding a small molecule in-
hibitor by rational design or HT screening remains difficult.
A large part of the challenge is that compounds in exist-
ing libraries are structurally biased towards classical targets
for which they were initially developed, such as enzymes,
ion channels and G-protein coupled receptors. Nonetheless,
small molecule inhibitors for several protein–protein inter-
actions have been identified, e.g. disrupting the interaction
between human p53 with human MDM2 or between the hu-
man papillomavirus (HPV) E1 and E2 proteins [260,264]. The
available examples indicate that inhibitors of protein interac-
tions, indeed, do not exhibit structural similarity with other
classes of small molecule inhibitors [260]. Thus, it is likely
that protein–protein interaction “inhibitors are likely more iso-
lated in chemical space” [260]. In addition to expanding ex-
isting chemical libraries, availability of reliable information
on protein interactions and their role in physiological and
pathophysiological processes is critical for the development
protein–protein interaction-directed therapeutics.
10 Summary and outlook
In the 20th century, the basic principles of molecular bio-
logical regulation have been discovered and described. Dur-
ing this time, physical interactions among proteins were
found to mediate a vast range of regulatory functions in es-
sentially every biological process. In the first decade of the
21st century, insights into the global structure of protein–
protein interaction networks were gained, and the relation-
ship between genetics and the physical interactions of the
encoded genes is emerging. However, big challenges re-
main. As we have discussed, the biochemical relationships
between proteins and the functional consequences of phys-
ical interactions are profoundly more complicated than our
current understanding for the vast majority of proteins. Sim-
ilarly our bioinformatic tools—ranging from databases to
quantitative dynamic process models—need to become more
suited to represent and productively analyze this biochemical
complexity, once it is experimentally accessible on a larger
scale.
Network analysis is well suited to deal with complexity
and aims to detect large-scale organizational principles of
complex systems by intentionally ignoring, e.g. mechanis-
tic biochemical, details [265, 266]. Even though it is a young
field, as briefly discussed above, network analysis has already
contributed important insights to the understanding of bi-
ological systems (see also [211, 267]). A central question of
biological research, especially in our modern era, is how ge-
netic variation leads to desirable and pathologic phenotypes.
Genetics, although its tools have become more powerful than
ever before in the history of biology, can only provide part of
the answer. Genetic information needs to be complemented
with wiring diagrams and maps of how the encoded proteins
functionally and physically interact with each other [268]. As

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
has been shown for both human disease and plants, there is
a great potential for even simple variation of primary protein
sequence, e.g. due to natural genetic variation or pathologic
or experimental selection, to modulate interactions qualita-
tively (all or none) or quantitatively (interaction strength) and
thereby affect phenotypes [263, 269–271]. Thus knowledge
of interaction wiring not only feeds global network analy-
sis, but can provide the foundation for an understanding of
the specific molecular mechanisms underlying pathologic or
desirable phenotypes. However, at the same time, interac-
tion maps need to become more comprehensive to optimally
complement modern genomic data, and interaction analysis
technologies need to become more versatile to probe protein–
protein interactions under different physiological conditions.
In order to overcome current limitations, new technologies
for protein–protein interaction analysis will be required. The
articles in this issue of PROTEOMICS provide an overview
over state-of-the-art technologies and ongoing technology de-
velopment for analysis of protein–protein interactions and
their dynamic regulation, and for strategies toward obtaining
high-quality interactome maps of increasing completeness.
The authors have declared no conflict of interest.
11 References
[1] Durbin, R. M., Abecasis, G. R., Altshuler, D. L., Auton, A.
et al., A map of human genome variation from population-
scale sequencing. Nature 2010, 467, 1061–1073.
[2] Vickery, H. B., Origin of the word protein. Nature 1951, 168,
244.
[3] Keith, L. M., Louis Pasteur (1822–1895) — chance and the
prepared mind. Trends Biotechnol. 1995, 13, 511–515.
[4] Hein, G. E., The Liebig-Pasteur controversy: vitality without
vitalism. J. Chem. Edu. 1961, 38, 614.
[5] Payen and Persoz, Mémoire sur la diastase, et les prin-
cipaux produits de ses réactions. Ann. de Chimie et de
Physique 1933, 53, 73–92.
[6] Florkin, M., Discovery of pepsin by Theodor Schwann. Re-
vue Medicale De Liege 1957, 12, 139–144.
[7] Kühne, W., Ueber das Trypsin. Verhandlungen des
naturhistorisch-medicinischen Vereins zu Heidelberg 1876,
1, 194–198.
[8] Hedin, S. G., Trypsin and antitrypsin. Biochem. J. 1906, 1,
474–483.
[9] Northrop, J. H., The inactivation of trypsin : II. the equilib-
rium between trypsin and the inhibiting substance formed
by its action on proteins. J. Gen. Physiol. 1922, 4, 245–260.
[10] Sumner, J. B., The isolation and crystallization of the en-
zyme urease. Preliminary paper. J. Biol. Chem. 1926, 69,
435–441.
[11] Bradberry, S., Ricin and abrin. 2007, 35, 576–577.
[12] Ehrlich, P., Experimentelle Untersuchungen über Immu-
nität. II. Ueber Abrin. Deutsche Medizinische Wochenschrift
1891, 17, 1218–1219.
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
[13] Umemura, R., The relation between the absorption of anti-
bodies and the isolated protein bodies. J. Immunol. 1921,
6, 205–222.
[14] Buchner, E., Alkoholische Gärung ohne Hefezellen
(Vorläufige Mitteilung). Berichte der Deutschen Chemis-
chen. Gesellschaft 1897, 30, 117–124.
[15] Kastle, J. H., On the vital activity of the enzymes. Science
1901, 13, 765–771.
[16] Loeb, J., The proteins and colloid chemistry. Science 1920,
52, 449–456.
[17] Loew, O., On the chemical nature of enzymes. Science
(New York, N.Y.) 1899, 10, 955–961.
[18] Miller, E. R., The chemical nature of enzymes. Science
1929, 70, 356–357.
[19] Svedberg, T., Chirnoaga. E., The molecular weight
of hemocyanin. J. Am. Chem. Soc. 1928, 50, 1399–
1411.
[20] Svedberg, T., Mass and size of protein molecules. Nature
1929, 123, 871.
[21] Inga, B., Eriksson, Q., Svedberg, T., The molecular weights
and pH-stability regions of the hemocyanins. Biol. Bull.
1936, 71, 498–547.
[22] van Holde, K. E., Reflections on a century of protein chem-
istry. Biophys. Chem. 2003, 100, 71–79.
[23] Bergmann, M., The structure of proteins in relation to bio-
logical problems. Chem. Rev. 1938, 22, 423–435.
[24] Sanger, F., Tuppy, H., The amino-acid sequence in the
phenylalanyl chain of insulin .1. The identification of lower
peptides from partial hydrolysates. Biochem. J. 1951, 49,
463–481.
[25] Sanger, F., Tuppy, H., The amino-acid sequence in the
phenylalanyl chain of insulin .2. The investigation of pep-
tides from enzymic hydrolysates. Biochem. J. 1951, 49,
481–490.
[26] Sanger, F., Some peptides from insulin. Nature 1948, 162,
491–492.
[27] Sanger, F., Some chemical investigations on the structure
of insulin. Cold Spring Harb. Symp. Quant. Biol. 1950, 14,
153–160.
[28] Tiselius, A., A new apparatus for electrophoretic analysis
of colloidal mixtures. Trans. Faraday Soc. 1937, 33, 0524–
0530.
[29] Tiselius, A., Adsorption analysis of amino acid mixtures.
Adv. Protein Chem. 1947, 3, 67–93.
[30] Consden, R., Gordon, A. H., Martin, A. J. P., Qualitative
analysis of proteins: a partition chromatographic method
using paper. Biochem. J. 1944, 38, 224–232.
[31] Sanger, F., The arrangement of amino acids in proteins.
Adv. Protein Chem. 1952, 7, 1–67.
[32] Sanger, F., Thompson, E. O. P., The amino-acid sequence
in the glycyl chain of insulin .1. The identification of lower
peptides from partial hydrolysates. Biochem. J. 1953, 53,
353–366.
[33] Sanger, F., Thompson, E. O. P., The amino-acid sequence in
the glycyl chain of insulin .2. The investigation of peptides

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1491
from enzymic hydrolysates. Biochem. J. 1953, 53, 366–
374.
[34] Hauptman, H. A., History of x-ray crystallography.
Chemometr. Intell. Lab. Sys. 1991, 10, 13–18.
[35] Kendrew, J. C., Structure and function in myoglobin and
other proteins. Fed. Proc. 1959, 18, 740–751.
[36] Perutz, M. F., Structure of hemoglobin. Brookhaven Symp.
Biol. 1960, 13, 165–183.
[37] Monod, J., Wyman, J., Changeux, J. P., On the nature of
allosteric transitions: a plausible model. J. Mol. Biol. 1965,
12, 88–118.
[38] Perutz, M. F., Fersht, A. R., Simon, S. R., Roberts, G. C.
K., Influence of globin structure on state of heme .2. Al-
losteric transitions in methemoglobin. Biochemistry 1974,
13, 2174–2186.
[39] Watson, J. D., Crick, F. H. C., Molecular structure of nucleic
acids - a structure for deoxyribose nucleic acid. Nature
1953, 171, 737–738.
[40] Aristotle (Peck, A. L., Foster, E. S., translators), Parts of
Animals, Movement of Animals, Progression of Animals.,
Loeb Classical Library No. 323, 1937.
[41] Norris, R., Researches on muscular irritability and the re-
lations which exist between muscle, nerve and blood.
J. Anat. Physiol. 1867, 1, 217–236.
[42] von Muralt, L., Edsall, J. T., Double refraction of myosin and
its relation to the structure of the muscle fibre. Transac.
Faraday Soc. 1930, 26, 0837–0863.
[43] Engelhardt, W. A., Ljubimowa, M. N., Myosine and
adenosinetriphosphatase. Nature 1939, 144, 668–669.
[44] Bailey, K., Myosin and adenosine triphosphatase.
Biochem. J. 1942, 36, 121–139.
[45] Needham, D. M., The adenosine triphosphatase activity of
myosin preparations. Biochem. J. 1942, 36, 113–120.
[46] Dainty, M., Kleinzeller, A., Lawrence, A. S. C., Miall, M.
et al., Studies on the anomalous viscosity and flow-
birefringence of protein solutions iii. Changes in these
properties of myosin solutions in relation to adenosine
triphosphatase and muscular contraction. J. Gen. Physiol.
1944, 27, 355–399.
[47] Banga, I., Szent-Györgyi A. G., Preparation and properties
of myosin A and B. Stud. Inst. Med. Chem. Univ. Szeged.
1942, I:5–15.
[48] Guba, F., Observations on myosin and actomyosin. Stud.
Inst. Med. Chem. Univ. Szeged. 1943, III:40–45.
[49] Jakus, M. A., Hall, C. E., Studies of actin and myosin.
J. Biol. Chem. 1947, 167, 705–714.
[50] Kesztyus, L., Nikodemusz, S., Szilagyi, T., Antigenic activity
of myosin and actin. Nature 1949, 163, 136.
[51] Szent-Gyorgyi, A. G., Milestone in physiology - the early
history of the biochemistry of muscle contraction. J. Gen.
Physiol. 2004, 123, 631–641.
[52] Straub, F. B., Actin. Stud. Inst. Med. Chem. Univ. Szeged
1942, II:3–15.
[53] Straub, F. B., Actin, II. Stud. Inst. Med. Chem. Univ. Szeged
1943, III:23–37.
www.proteomics-journal.com
1492 P. Braun and A.-C. Gingras
[54] Straub, F. B., Note on the work of f. Bruno Straub con-
cerning ‘adenosine triphosphate. The functional group of
actin’. Biochim. Biophys. Acta 1989, 1000, 179.
[55] Straub, F. B., Feuer, G., [adenosine triphosphate, the func-
tional group of actin]. Kiserl Orvostud 1950, 2, 141–151.
[56] Straub, F. B., Feuer, G., Adenosinetriphosphate. The func-
tional group of actin. 1950. Biochim. Biophys. Acta 1989,
1000, 180–195.
[57] Huxley, H. E., The mechanism of muscular contraction.
Clin. Orthop. Related Res. 2002, S6–S17.
[58] Huxley, H. E., Mechanism of muscular contraction. Science
1969, 164, 1356–1366.
[59] Fletcher, D. A., Mullins, D., Cell mechanics and the cy-
toskeleton. Nature 2010, 463, 485–492.
[60] Beadle, G. W., Tatum, E. L., Genetic control of biochemical
reactions in neurospora. Proc. Natl. Acad. Sci. USA 1941,
27, 499–506.
[61] Horowitz, N. H., Beadle, George, Wells (1903-1989) - obit-
uary. Genetics 1990, 124, 1–6.
[62] Sanadi, D. R., Littlefield, J. W., Studies on alpha-
ketoglutaric oxidase .2. Purification and properties. J. Biol.
Chem. 1952, 197, 851–862.
[63] Koike, M., Reed, L. J., Carroll, W. R., Alpha-keto acid dehy-
drogenation complexes .1. Purification and properties of
pyruvate and alpha-ketoglutarate dehydrogenation com-
plexes of Escherichia coli. J. Biol. Chem. 1960, 235, 1924–
1930.
[64] Hayakawa, T., Muta, H., Hirashima, M., Ide, S. et al., Isola-
tion and properties of pyruvate and ␣-ketoglutarate dehy-
drogenation complexes from pig heart muscle. Biochem.
Biophys. Res. Commun. 1964, 17, 51–56.
[65] Frieden, C., Protein-protein interaction and enzymatic ac-
tivity. Annu. Rev. Biochem. 1971, 40, 653–696.
[66] Arnold, H., Pette, D., Binding of aldolase and triosephos-
phate dehydrogenase to F-actin and modification of cat-
alytic properties of aldolase. Eur. J. Biochem. 1970, 15,
360–366.
[67] Crick, F. H. C., Orgel, L. E., The theory of inter-allelic com-
plementation. J. Mol. Biol. 1964, 8, 161–165.
[68] McGavin, S., Interallelic complementation and allostery. J.
Mol. Biol. 1968, 37, 239–242.
[69] Fischer, E. H., Phosphorylase and the origin of reversible
protein phosphorylation. Biol. Chem. 2010, 391, 131–137.
[70] Cori, G. T., Cori, C. F., The enzymatic conversion of
phosphorylase-a to phosphorylase-b. J. Biol. Chem. 1945,
158, 321–332.
[71] Cori, G. T., Green, A. A., Crystalline muscle phosphorylase
ii. Prosthetic group. J. Biol. Chem. 1943, 151, 31–38.
[72] Barford, D., Hu, S. H., Johnson, L. N., Structural mecha-
nism for glycogen-phosphorylase control by phosphoryla-
tion and AMP. J. Mol. Biol. 1991, 218, 233–260.
[73] Fischer, E. H., Krebs, E. G., Conversion of phosphorylase-b
to phosphorylase-a in muscle extracts. J. Biol. Chem. 1955,
216, 121–132.
[74] Krebs, E. G., Fischer, E. H., The phosphorylase b to a con-

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
verting enzyme of rabbit skeletal muscle. Biochim. Bio-
phys. Acta 1956, 20, 150–157.
[75] Graves, D. J., Fischer, E. H., Krebs, E. G., Specificity stud-
ies on muscle phosphorylase phosphatase. J. Biol. Chem.
1960, 235, 805–809.
[76] Brandt, H., Capulong, Z. L., Lee, E. C., Purification and prop-
erties of rabbit liver phosphorylase phosphatase. J. Biol.
Chem. 1975, 250, 8038–8044.
[77] Davoren, P. R., Sutherland, E. W., The effect of L-
epinephrine and other agents on the synthesis and re-
lease of adenosine 3’,5’-phosphate by whole pigeon ery-
throcytes. J. Biol. Chem. 1963, 238, 3009–3015.
[78] Rall, T. W., Sutherland, E. W., Formation of a cyclic adenine
ribonucleotide by tissue particles. J. Biol. Chem. 1958, 232,
1065–1076.
[79] Walsh, D. A., Perkins, J. P., Krebs, E. G., An adeno-
sine 3’,5’-monophosphate-dependant protein kinase from
rabbit skeletal muscle. J. Biol. Chem. 1968, 243, 3763–
3765.
[80] Sutherland, E. W., Robison, G. A., Role of cyclic AMP in
control of carbohydrate metabolism. Diabetes 1969, 18,
797–819.
[81] Taylor, S. S., cAMP-dependent protein kinase. Model
for an enzyme family. J. Biol. Chem. 1989, 264, 8443–
8446.
[82] Corbin, J. D., Keely, S. L., Soderling, T. R., Park, C. R.,
Hormonal regulation of adenosine 3’,5’-monophosphate-
dependent protein kinase. Adv. Cyclic Nucl. Res. 1975, 5,
265–279.
[83] Beavo, J. A., Bechtel, P. J., Krebs, E. G., Mechanisms of
control for cAMP-dependent protein kinase from skeletal
muscle. Adv. Cyclic Nucl. Res. 1975, 5, 241–251.
[84] Hubbard, M. J., Cohen, P., On target with a new mecha-
nism for the regulation of protein-phosphorylation. Trends
Biochem. Sci. 1993, 18, 172–177.
[85] Lefkowitz, R. J., Historical review: a brief history and per-
sonal retrospective of seven-transmembrane receptors.
Trends Pharmacol. Sci. 2004, 25, 413–422.
[86] Rang, H. P., The receptor concept: pharmacology’s big idea.
Brit. J. Pharmacol. 2006, 147, S9–S16.
[87] Changeux, J. P., Allosteric receptors: from electric organ to
cognition. Annual Review of Pharmacology and Toxicol-
ogy, Annual Reviews, Palo Alto 2010, pp. 1–38.
[88] Cuatreca, P., Insulin-receptor interactions in adipose tissue
cells: direct measurement and properties. Proc. Natl. Acad.
Sci. USA 1971, 68, 1264–1268.
[89] Carpenter, G., Cohen, S., I125 labeled human epidermal
growth-factor - binding, internalization, and degradation
in human fibroblasts. J. Cell Biol. 1976, 71, 159–171.
[90] Fambroug, D. M., Hartzell, H. C., Acetylcholine receptors -
number and distribution at neuromuscular junctions in rat
diaphragm. Science 1972, 176, 189–191.
[91] Lefkowitz, R. J., Roth, J., Pricer, W., Pastan, I., ACTH recep-
tors in the adrenal: specific binding of ACTH-125i and its
relation to adenyl cyclase. Proc. Natl. Acad. Sci. 1970, 65,
745–752.
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
[92] Miledi, R., Molinoff, P., Potter, L. T., Isolation of cholinergic
receptor protein of torpedo electric tissue. Nature 1971,
229, 554–557.
[93] Patrick, J., Lindstro, J., Autoimmune response to acetyl-
choline receptor. Science 1973, 180, 871–872.
[94] Sugiyama, H., Benda, P., Meunier, J. C., Changeux, J. P., Im-
munological characterization of cholinergic receptor pro-
tein from electrophorus-electricus. FEBS Lett. 1973, 35,
124–128.
[95] Hucho, F., Changeux, J.-P., Molecular weight and quater-
nary structure of the cholinergic receptor protein extracted
by detergents from electrophorus electricus electric tissue.
FEBS Lett. 1973, 38, 11–15.
[96] Milligan, G., Kostenis, E., Heterotrimeric G-proteins: a short
history. Brit. J. Pharmacol. 2006, 147, S46–S55.
[97] Dulbecco, R., Vogt, M., Plaque formation and isolation of
pure lines with poliomyelitis viruses. J. Exp. Med. 1954, 99,
167–182.
[98] Laemmli, U. K., Cleavage of structural proteins during as-
sembly of head of bacteriophage-T4. Nature 1970, 227,
680–685.
[99] Kohler, G., Milstein, C., Continuous cultures of fused cells
secreting antibody of predefined specificity. Nature 1975,
256, 495–497.
[100] Gubler, U., Hoffman, B. J., A simple and very efficient
method for generating cDNA libraries. Gene 1983, 25, 263–
269.
[101] Okayama, H., Berg, P., High-efficiency cloning of full-length
cDNA. Mol. Cell. Biol. 1982, 2, 161–170.
[102] Southern, E. M., Detection of specific sequences among
DNA fragments separated by gel-electrophoresis. J. Mol.
Biol. 1975, 98, 503–517.
[103] Sanger, F., Nicklen, S., Coulson, A. R., DNA sequencing
with chain-terminating inhibitors. Proc. Natl. Acad. Sci.
USA 1977, 74, 5463–5467.
[104] Meselson, M., Yuan, R., DNA restriction enzyme from
E. coli. Nature 1968, 217, 1110–1114.
[105] Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J. et al.,
Primer-directed enzymatic amplification of DNA with a
thermostable DNA-polymerase. Science 1988, 239, 487–
491.
[106] Wagner, R. P., Rudolph Virchow and the genetic basis of
somatic ecology. Genetics 1999, 151, 917–920.
[107] Hartwell, L. H., Culotti, J., Pringle, J. R., Reid, B. J., Genetic-
control of cell-division cycle in yeast. Science 1974, 183,
46–51.
[108] Nurse, P., Thuriaux, P., Nasmyth, K., Genetic-control of
cell-division cycle in fission yeast Schizosaccharomyces
pombe. Mol. Gen. Genet. 1976, 146, 167–178.
[109] Durkacz, B., Beach, D., Hayles, J., Nurse, P., The fission
yeast-cell cycle control gene cdc2 - structure of the cdc2
region. Mol. Gen. Genet. 1985, 201, 543–545.
[110] Simanis, V., Nurse, P., The cell-cycle control gene
cdc2+ of fission yeast encodes a protein-kinase po-
tentially regulated by phosphorylation. Cell 1986, 45,
261–268.

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1493
[111] Masui, Y., Markert, C. L., Cytoplasmic control of nuclear
behavior during meiotic maturation of frog oocytes. J. Exp.
Zool. 1971, 177, 129-&.
[112] Wasserman, W. J., Masui, Y., Cytoplasmic factor promoting
oocyte maturation - its extraction and preliminary charac-
terization. Science 1976, 191, 1266–1268.
[113] Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D. et al.,
Cyclin - a protein specified by maternal messenger-RNA in
sea-urchin eggs that is destroyed at each cleavage division.
Cell 1983, 33, 389–396.
[114] Pines, J., Hunt, T., Molecular-cloning and characterization
of the messenger-RNA for cyclin from sea-urchin eggs.
Embo J. 1987, 6, 2987–2995.
[115] Nurse, P., Masui, Y., Hartwell, L., Understanding the cell
cycle. Nat. Med. 1998, 4, 1103–1106.
[116] Lee, M. G., Nurse, P., Complementation used to clone a
human homolog of the fission yeast-cell cycle control gene
cdc2. Nature 1987, 327, 31–35.
[117] Gautier, J., Norbury, C., Lohka, M., Nurse, P. et al., Puri-
fied maturation-promoting factor contains the product of
a Xenopus homolog of the fission yeast-cell cycle control
gene cdc2+. Cell 1988, 54, 433–439.
[118] Labbe, J. C., Lee, M. G., Nurse, P., Picard, A. et al., Activa-
tion at M-phase of a protein-kinase encoded by a starfish
homolog of the cell-cycle control gene cdc2+. Nature 1988,
335, 251–254.
[119] Gautier, J., Minshull, J., Lohka, M., Glotzer, M. et al., Cy-
clin is a component of maturation-promoting factor from
Xenopus. Cell 1990, 60, 487–494.
[120] Pines, J., Hunter, T., Isolation of a human cyclin CDNA - evi-
dence for cyclin messenger-RNA and protein-regulation in
the cell-cycle and for interaction with p34-cdc2. Cell 1989,
58, 833–846.
[121] Solomon, M. J., Glotzer, M., Lee, T. H., Philippe, M. et al.,
Cyclin activation of p34cdc2. Cell 1990, 63, 1013–1024.
[122] Crawford, L. V., Lane, D. P., Denhardt, D. T., Harlow, E. E.
et al., Characterization of the complex between sv40 large
T-antigen and the 53k host protein in transformed mouse
cells. Cold Spring Harb. Symp. Quant. Biol. 1979, 44, 179–
187.
[123] Lane, D. P., Crawford, L. V., T-antigen is bound to a host
protein in SV40-transformed cells. Nature 1979, 278, 261–
263.
[124] Brugge, J. S., Erikson, R. L., Identification of a
transformation-specific antigen-induced by an avian-
sarcoma virus. Nature 1977, 269, 346–348.
[125] Levinson, A. D., Oppermann, H., Levintow, L., Varmus,
H. E., et al., Evidence that transforming gene of avian-
sarcoma virus encodes a protein-kinase associated with
a phosphoprotein. Cell 1978, 15, 561–572.
[126] Collett, M. S., Erikson, R. L., Protein-kinase activity associ-
ated with avian-sarcoma virus src gene product. Proc. Natl.
Acad. Sci. USA 1978, 75, 2021–2024.
[127] Eckhart, W., Hutchinson, M. A., Hunter, T., Activity phos-
phorylating tyrosine in polyoma T-antigen immunoprecip-
itates. Cell 1979, 18, 925–933.
www.proteomics-journal.com
1494 P. Braun and A.-C. Gingras
[128] Ushiro, H., Cohen, S., Identification of phosphotyrosine as
a product of epidermal growth factor-activated protein-
kinase in A431 cell membranes. J. Biol. Chem. 1980, 255,
8363–8365.
[129] Konopka, J. B., Watanabe, S. M., Witte, O. N., An alter-
ation of the human c-abl protein in K562 leukemia-cells
unmasks associated tyrosine kinase-activity. Cell 1984, 37,
1035–1042.
[130] Gribskov, M., McLachlan, A. D., Eisenberg, D., Profile anal-
ysis - detection of distantly related proteins. Proc. Natl.
Acad. Sci. USA 1987, 84, 4355–4358.
[131] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., et al.,
Basic local alignment search tool. J. Mol. Biol. 1990, 215,
403–410.
[132] Sadowski, I., Stone, J. C., Pawson, T., A noncatalytic do-
main conserved among cytoplasmic protein-tyrosine ki-
nases modifies the kinase function and transforming activ-
ity of Fujinami sarcoma-virus p130gag-fps. Mol. Cell. Biol.
1986, 6, 4396–4408.
[133] Anderson, D., Koch, C. A., Grey, L., Ellis, C. et al., Binding of
SH2 domains of Phospholipase-C-gamma-1, GAP, and Src
to activated growth-factor receptors. Science 1990, 250,
979–982.
[134] Schlessinger, J., Cell signaling by receptor tyrosine ki-
nases. Cell 2000, 103, 211–225.
[135] Pawson, T., Protein modules and signaling networks.
Nature 1995, 373, 573–580.
[136] Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend,
S. H. et al., Association between an oncogene and an
anti-oncogene - the adenovirus E1A proteins bind to
the retinoblastoma gene-product. Nature 1988, 334, 124–
129.
[137] Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R.
et al., Chromosome-17 deletions and p53 gene-mutations
in colorectal carcinomas. Science 1989, 244, 217–221.
[138] Oltvai, Z. N., Milliman, C. L., Korsmeyer, S. J., Bcl-2 het-
erodimerizes in vivo with a conserved homolog, bax, that
accelerates programmed cell-death. Cell 1993, 74, 609–619.
[139] Colledge, M., Scott, J. D., AKAPs: from structure to func-
tion. Trends in Cell Biol. 1999, 9, 216–221.
[140] Taylor, S. S., Buechler, J. A., Yonemoto, W., cAMP-
dependent protein-kinase - framework for a diverse family
of regulatory enzymes. Annu. Rev. Biochem. 1990, 59, 971–
1005.
[141] Smith, F. D., Scott, J. D., Anchored cAMP signaling: onward
and upward - a short history of compartmentalized cAMP
signal transduction. Eur. J. Cell Biol. 2006, 85, 585–592.
[142] Buxton, I. L. O., Brunton, L. L., Compartments of cyclic-
AMP and protein-kinase in mammalian cardiomyocytes.
J. Biol. Chem. 1983, 258, 233–239.
[143] Bregman, D. B., Bhattacharyya, N., Rubin, C. S., High-
affinity binding-protein for the regulatory subunit of camp-
dependent protein kinase II-b - cloning, characterization,
and expression of cdnas for rat-brain p150. J. Biol. Chem.
1989, 264, 4648–4656.
[144] Rosenmund, C., Carr, D. W., Bergeson, S. E., Nilaver,
G. et al., Anchoring of protein-kinase-A is required for

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
modulation of AMPA/kainate receptors on hippocampal-
neurons. Nature 1994, 368, 853–856.
[145] Mor, A., Philips, M. R., Compartmentalized Ras/MAPK sig-
naling. Annual Review of Immunology, Annual Reviews,
Palo Alto 2006, pp. 771–800.
[146] Jacob, F., Ullmann, A., Monod, J., Délétions fusionnant
l’opéron lactose et un opéron purine chez Escherichia coli.
J. Mol. Biol. 1965, 13, 704–719.
[147] Shuman, H. A., Silhavy, T. J., Beckwith, J. R., Labeling of
proteins with beta-galactosidase by gene fusion - iden-
tification of a cytoplasmic membrane component of the
Escherichia coli maltose transport-system. J. Biol. Chem.
1980, 255, 168–174.
[148] Germino, J., Bastia, D., Rapid purification of a cloned gene-
product by genetic fusion and site-specific proteolysis.
Proc. Natl. Acad. Sci. USA-Biolog. Sci. 1984, 81, 4692–
4696.
[149] Smith, D. B., Johnson, K. S., Single-step purification of
polypeptides expressed in escherichia-coli as fusions with
glutathione s-transferase. Gene 1988, 67, 31–40.
[150] Hammarberg, B., Nygren, P. A., Holmgren, E., Elmblad,
A. et al., Dual affinity fusion approach and its use to
express recombinant human insulin-like growth factor-ii.
Proc. Natl. Acad. Sci. USA 1989, 86, 4367–4371.
[151] Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M. et al., A
generic protein purification method for protein complex
characterization and proteome exploration. Nat. Biotech-
nol. 1999, 17, 1030–1032.
[152] Puig, O., Caspary, F., Rigaut, G., Rutz, B. et al., The tandem
affinity purification (TAP) method: a general procedure
of protein complex purification. Methods 2001, 24, 218–
229.
[153] Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. et al., Affin-
ity fusion strategies for detection, purification, and immo-
bilization of recombinant proteins. Protein Expres. Purif.
1997, 11, 1–16.
[154] Niman, H. L., Houghten, R. A., Walker, L. E., Reisfeld, R. A.
et al., Generation of protein-reactive antibodies by short
peptides is an event of high-frequency - implications for
the structural basis of immune recognition. Proc. Natl.
Acad. Sci. USA-Biolog. Sci. 1983, 80, 4949–4953.
[155] Edman, P., Begg, G., A protein sequenator. Eur. J. Biochem.
1967, 1, 80–91.
[156] Karas, M., Hillenkamp, F., Laser desorption ionization of
proteins with molecular masses exceeding 10000 daltons.
Anal. Chem. 1988, 60, 2299–2301.
[157] Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. et al.,
Electrospray ionization for mass-spectrometry of large
biomolecules. Science 1989, 246, 64–71.
[158] Louris, J. N., Wright, L. G., Cooks, R. G., Schoen, A. E., New
scan modes accessed with a hybrid mass-spectrometer.
Anal. Chem. 1985, 57, 2918–2924.
[159] Aebersold, R., Mann, M., Mass spectrometry-based pro-
teomics. Nature 2003, 422, 198–207.
[160] Fields, S., Song, O., A novel genetic system to detect
protein-protein interactions. Nature 1989, 340, 245–246.
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
[161] Vojtek, A. B., Hollenberg, S. M., Cooper, J. A., Mammalian
ras interacts directly with the serine threonine kinase raf.
Cell 1993, 74, 205–214.
[162] Bartel, P. L., Roecklein, J. A., SenGupta, D., Fields, S., A
protein linkage map of Escherichia coli bacteriophage t7.
Nat. Genet. 1996, 12, 72–77.
[163] Petschnigg, J., Moe, O. W., Stagljar, I., Using yeast as a
model to study membrane proteins. Curr. Opin. Nephrol.
Hy. 2011, 20, 425–432.
[164] Michnick, S. W., Ear, P. H., Landry, C., Malleshaiah, M. K.
et al., in: Weissman, J., Guthrie, C., Fink, G. R. (Eds.), Meth-
ods in Enzymology, Vol 470: Guide to Yeast Genetics: Func-
tional Genomics, Proteomics, and Other Systems Analysis,
2nd Edition, Elsevier Academic Press Inc., San Diego 2010,
pp. 335–368.
[165] Lievens, S., Eyckerman, S., Lemmens, I., Tavernier, J.,
Large-scale protein interactome mapping: strategies and
opportunities. Exp. Rev. Proteomics 2010, 7, 679–690.
[166] Vidal, M., Legrain, P., Yeast forward and reverse ‘n’-hybrid
systems. Nucleic Acids Res. 1999, 27, 919–929.
[167] Braun, P., Interactome mapping for analysis of com-
plex phenotypes: insights from benchmarking bi-
nary interaction assays. Proteomics 2012, 12, doi:
10.1002/pmic.201100598.
[168] Roberts, L., Davenport, R. J., Pennisi, E., Marshall, E., A
history of the human genome project. Science (New York,
N.Y.) 2001, 291, 1195.
[169] Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J.
L. et al., Gateway recombinational cloning: application to
the cloning of large numbers of open reading frames or
ORFeomes. Methods Enzymol. 2000, 328, 575–592.
[170] Reboul, J., Vaglio, P., Rual, J. F., Lamesch, P. et al.,
C. elegans ORFeome version 1.1: experimental verification
of the genome annotation and resource for proteome-scale
protein expression. Nat. Genet. 2003, 34, 35–41.
[171] Lamesch, P., Li, N., Milstein, S., Fan, C. et al., HORFeome
v3.1: a resource of human open reading frames represent-
ing over 10,000 human genes. Genomics 2007, 89, 307–
315.
[172] Hilson, P., Cloned sequence repertoires for small- and
large-scale biology. Trends Plant Sci. 2006, 11, 133–141.
[173] Yamada, K., Lim, J., Dale, J. M., Chen, H. M. et al., Empir-
ical analysis of transcriptional activity in the Arabidopsis
genome. Science 2003, 302, 842–846.
[174] Rolfs, A., Hu, Y., Ebert, L., Hoffmann, D. et al., A biomedi-
cally enriched collection of 7000 human ORF clones. PLoS
One 2008, 3, e1528. DOI: 10.1371/journal.pone.0001528
[175] Yang, X., Boehm, J. S., Yang, X., Salehi-Ashtiani, K. et al., A
public genome-scale lentiviral expression library of human
ORFs. Nat. Methods 2011, 8, U659–U680.
[176] Yu, C., Wan, K. H., Hammonds, A. S., Stapleton, M. et al.,
in: Wu, C. J. (Ed.), Protein Microarray for Disease Analysis:
Methods and Protocols, Humana Press Inc., Totowa, NJ
2011, pp. 257–272.
[177] Colwill, K., Wells, C. D., Elder, K., Goudreault, M. et al.,
Modification of the creator recombination system for

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1495
proteomics applications - improved expression by addi-
tion of splice sites. BMC Biotechnol. 2006, 6, 13. DOI:
10.1186/1472-6750-6-13.
[178] Park, J., Labaer, J., UNIT 3.20 Recombinational cloning.
Curr. Protocols Mol. Biol./edited by Frederick M. Ausubel
. . . [et al.] 2006, Chapter 3.
[179] Nilsson, S., Svedberg, T. M., Pettersson, J., Bjorefors, T. F.
et al., Evaluations of the stability of sheathless electrospray
ionization mass spectrometry emitters using electrochem-
ical techniques. Anal. Chem. 2001, 73, 4607–4616.
[180] Oliver, S., Guilt-by-association goes global. Nature 2000,
403, 601–603.
[181] Uetz, P., Giot, L., Cagney, G., Mansfield, T., A compre-
hensive analysis of protein-protein interactions in Saccha-
romyces cerevisiae. Nature 2000, 403, 623–627.
[182] Walhout, A. J., Sordella, R., Lu, X., Hartley, J. L. et al.,
Protein interaction mapping in C. elegans using proteins
involved in vulval development. Science 2000, 287, 116–
122.
[183] Ito, T., Tashiro, K., Muta, S., Ozawa, R. et al., Toward
a protein-protein interaction map of the budding yeast:
a comprehensive system to examine two-hybrid interac-
tions in all possible combinations between the yeast pro-
teins. Proc. Natl. Acad. Sci. USA 2000, 97, 1143–1147.
[184] Ito, T., Chiba, T., Ozawa, R., Yoshida, M. et al., A compre-
hensive two-hybrid analysis to explore the yeast protein
interactome. Proc. Natl. Acad. Sci. USA 2001, 98, 4569–
4574.
[185] Gavin, A., Bosche, M., Krause, R., Grandi, P., Functional
organization of the yeast proteome by systematic analysis
of protein complexes. Nature 2002, 415, 141–147.
[186] Ho, Y., Gruhler, A., Heilbut, A., Bader, G., Systematic identi-
fication of protein complexes in Saccharomyces cerevisiae
by mass spectrometry. Nature 2002, 415, 180–183.
[187] Gavin, A. C., Aloy, P., Grandi, P., Krause, R. et al., Proteome
survey reveals modularity of the yeast cell machinery. Na-
ture 2006, 440, 631–636.
[188] Krogan, N. J., Cagney, G., Yu, H., Zhong, G. et al., Global
landscape of protein complexes in the yeast Saccha-
romyces cerevisiae. Nature 2006, 440, 637–643.
[189] Giot, L., Bader, J., Brouwer, C., Chaudhuri, A., A protein in-
teraction map of Drosophila melanogaster. Science 2003,
302, 1727–1736.
[190] Li, S., Armstrong, C. M., Bertin, N., Ge, H. et al., A map
of the interactome network of the metazoan C. elegans.
Science 2004, 303, 540–543.
[191] Rual, J. F., Venkatesan, K., Hao, T., Hirozane-Kishikawa,
T. et al., Towards a proteome-scale map of the human
protein-protein interaction network. Nature 2005, 437,
1173–1178.
[192] Stelzl, U., Worm, U., Lalowski, M., Haenig, C. et al., A hu-
man protein-protein interaction network: a resource for
annotating the proteome. Cell 2005, 122, 957–968.
[193] Arabidopsis Interactome Mapping Consortium, Evidence
for network evolution in an Arabidopsis interactome map.
Science 2011, 333, 601–607.
www.proteomics-journal.com
1496 P. Braun and A.-C. Gingras
[194] Sato, S., Shimoda, Y., Muraki, A., Kohara, M. et al., A large-
scale protein protein interaction analysis in Synechocystis
sp. Pcc6803. DNA Res. 2007, 14, 207–216.
[195] Shimoda, Y., Shinpo, S., Kohara, M., Nakamura, Y. et al., A
large scale analysis of protein-protein interactions in the
nitrogen-fixing bacterium Mesorhizobium loti. DNA Res.
2008, 15, 13–23.
[196] Tarassov, K., Messier, V., Landry, C. R., Radinovic, S. et al.,
An in vivo map of the yeast protein interactome. Science
2008, 320, 1465–1470.
[197] Miller, J. P., Lo, R. S., Ben-Hur, A., Desmarais, C. et al.,
Large-scale identification of yeast integral membrane pro-
tein interactions. Proc. Natl. Acad. Sci. USA 2005, 102,
12123–12128.
[198] Lalonde, S., Sero, A., Pratelli, R. J., Pilot, G. et al., A
membrane protein/signaling protein interaction network
for Arabidopsis version ampv2. Front. Physiol. 2010, 1.
DOI: 10.3389/fphys.2010.00024.
[199] Huh, W. K., Falvo, J. V., Gerke, L. C., Carroll, A. S. et al.,
Global analysis of protein localization in budding yeast.
Nature 2003, 425, 686–691.
[200] Ghaemmaghami, S., Huh, W., Bower, K., Howson, R. W.
et al., Global analysis of protein expression in yeast. Nature
2003, 425, 737–741.
[201] Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang,
H. et al., Functional characterization of the S. cerevisiae
genome by gene deletion and parallel analysis. Science
1999, 285, 901–906.
[202] Giaever, G., Chu, A. M., Ni, L., Connelly, C. et al., Functional
profiling of the saccharomyces cerevisiae genome. Nature
2002, 418, 387–391.
[203] Tong, A. H. Y., Lesage, G., Bader, G. D., Ding, H. M. et al.,
Global mapping of the yeast genetic interaction network.
Science 2004, 303, 808–813.
[204] Zhu, H., Klemic, J. F., Chang, S., Bertone, P. et al., Analysis
of yeast protein kinases using protein chips. Nat. Genet.
2000, 26, 283–289.
[205] Ptacek, J., Devgan, G., Michaud, G., Zhu, H. et al., Global
analysis of protein phosphorylation in yeast. Nature 2005,
438, 679–684.
[206] Ge, H., Liu, Z. H., Church, G. M., Vidal, M., Correlation
between transcriptome and interactome mapping data
from saccharomyces cerevisiae. Nat. Genet. 2001, 29, 482–
486.
[207] Von Mering, C., Jensen, L. J., Snel, B., Hooper, S. D.
et al., String: known and predicted protein-protein asso-
ciations, integrated and transferred across organisms. Nu-
cleic Acids Res. 2005, 33, D433–D437.
[208] Jensen, L. J., Kuhn, M., Stark, M., Chaffron, S. et al., String
8-a global view on proteins and their functional interac-
tions in 630 organisms. Nucleic Acids Res. 2009, 37, D412–
D416.
[209] Jansen, R., Yu, H. Y., Greenbaum, D., Kluger, Y. et al., A
Bayesian networks approach for predicting protein-protein
interactions from genomic data. Science 2003, 302, 449–
453.

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
[210] Schwikowski, B., Uetz, P., Fields, S., A network of protein-
protein interactions in yeast. Nat. Biotechnol. 2000, 18,
1257–1261.
[211] Vidal, M., Cusick, M. E., Barabasi, A.-L., Interactome net-
works and human disease. Cell 2011, 144, 986–998.
[212] Barabasi, A. L., Bonabeau, E., Scale-free networks. Sci. Am.
2003, 288, 60–69.
[213] Albert, R., Jeong, H., Barabasi, A. L., Error and attack toler-
ance of complex networks. Nature 2000, 406, 378–382.
[214] Calderwood, M. A., Venkatesan, K., Xing, L., Chase, M. R.
et al., Epstein-Barr virus and virus human protein interac-
tion maps. Proc. Natl. Acad. Sci. USA 2007, 104, 7606–7611.
[215] Mukhtar, M. S., Carvunis, A.-R., Dreze, M., Epple, P. et al.,
Independently evolved virulence effectors converge onto
hubs in a plant immune system network. Science 2011,
333, 596–601.
[216] Friedel, C. C., Haas, J., Virus-host interactomes and global
models of virus-infected cells. Trends in Microbiol. 2011,
19, 501–508.
[217] Yang, H., Ke, Y., Wang, J., Tan, Y. et al., Insight into bacte-
rial virulence mechanisms against host immune response
via the Yersinia pestis-human protein-protein interaction
network. Infect. Immun. 2011, 79, 4413–4424.
[218] Navratil, V., de Chassey, B., Combe, C. R., Lotteau, V.,
When the human viral infectome and diseasome networks
collide: towards a systems biology platform for the ae-
tiology of human diseases. BMC Syst. Biol. 2011, 5, 13.
DOI:10.1186/1752-0509-5-13.
[219] Yu, H., Braun, P., Yildirim, M. A., Lemmens, I. et al., High-
quality binary protein interaction map of the yeast interac-
tome network. Science 2008, 322, 104–110.
[220] Goh, K. I., Cusick, M. E., Valle, D., Childs, B. et al., The
human disease network. Proc. Natl. Acad. Sci. USA 2007,
104, 8685–8690.
[221] Lim, J., Hao, T., Shaw, C., Patel, A. J. et al., A protein-
protein interaction network for human inherited ataxias
and disorders of Purkinje cell degeneration. Cell 2006, 125,
801–814.
[222] Soler-Lopez, M., Zanzoni, A., Lluis, R., Stelzl, U. et al., In-
teractome mapping suggests new mechanistic details un-
derlying Alzheimer’s disease. Genome Res. 2011, 21, 364–
376.
[223] Pujana, M. A., Han, J. D., Starita, L. M., Stevens, K. N. et al.,
Network modeling links breast cancer susceptibility and
centrosome dysfunction. Nat. Genet. 2007, 39, 1338–1349.
[224] Lage, K., Karlberg, E. O., Storling, Z. M., Olason, P. I. et al.,
A human phenome-interactome network of protein com-
plexes implicated in genetic disorders. Nat. Biotech. 2007,
25, 309–316.
[225] Breitkreutz, A., Choi, H., Sharom, J. R., Boucher, L. et al., A
global protein kinase and phosphatase interaction network
in yeast. Science 2010, 328, 1043–1046.
[226] Ulitsky, I., Shlomi, T., Kupiec, M., Shamir, R., From E-maps
to module maps: dissecting quantitative genetic interac-
tions using physical interactions. Mol. Syst. Biol. 2008, 4,
209.
www.proteomics-journal.com
Proteomics 2012, 12, 1478–1498
[227] Bandyopadhyay S., Kelley R., Krogan N. J., Ideker T.,
Functional maps of protein complexes from quantitative
genetic interaction data. PLoS Computat. Biol. 2008, 4,
e1000065. doi:10.1371/journal.pcbi.1000065.
[228] Hannum, G., Srivas, R., Guenole, A., van Attikum, H. et al.,
Genome-wide association data reveal a global map of ge-
netic interactions among protein complexes. PLoS Genet-
ics 2009, 5.
[229] Costanzo, M., Baryshnikova, A., Bellay, J., Kim, Y. et al., The
genetic landscape of a cell. Science 2010, 327, 425–431.
[230] Michaut, M., Baryshnikova, A., Costanzo, M., Myers, C. L.
et al., Protein complexes are central in the yeast genetic
landscape. PLoS Computat. Biol. 2011, 7, e1001092.
[231] Akula, N., Baranova, A., Seto, D., Solka, J. et al., A network-
based approach to prioritize results from genome-wide as-
sociation studies. PLoS One 2011, 6, e24220.
[232] Rossin, E. J., Lage, K., Raychaudhuri, S., Xavier, R. J.
et al., Proteins encoded in genomic regions associated with
immune-mediated disease physically interact and suggest
underlying biology. PLoS Genet. 2011, 7, e1001273.
[233] Taylor, I. W., Linding, R., Warde-Farley, D., Liu, Y. et al., Dy-
namic modularity in protein interaction networks predicts
breast cancer outcome. Nat. Biotechnol. 2009, 27, 199–204.
[234] Chuang, H.-Y., Lee, E., Liu, Y.-T., Lee, D. et al., Network-
based classification of breast cancer metastasis. Mol. Syst.
Biol. 2007, 3, 140.
[235] Barabasi, A.-L., Gulbahce, N., Loscalzo, J., Network
medicine: a network-based approach to human disease.
Nat. Rev. Genet. 2011, 12, 56–68.
[236] Venkatesan, K., Rual, J. F., Vazquez, A., Stelzl, U. et al., An
empirical framework for binary interactome mapping. Nat.
Methods 2009, 6, 83–90.
[237] Braun, P., Tasan, M., Dreze, M., Barrios-Rodiles, M. et al.,
An experimentally derived confidence score for binary
protein-protein interactions. Nat. Methods 2009, 6, 91–97.
[238] Chen, Y. C., Rajagopala, S. V., Stellberger, T., Uetz, P., Ex-
haustive benchmarking of the yeast two-hybrid system.
Nat. Methods 2010, 7, 667–668; author reply 668.
[239] Simonis, N., Rual, J. F., Carvunis, A. R., Tasan, M. et al.,
Empirically controlled mapping of the Caenorhabditis ele-
gans protein–protein interactome network. Nat. Methods
2009, 47–54.
[240] Stumpf, M. P. H., Thorne, T., de Silva, E., Stewart, R. et al.,
Estimating the size of the human interactome. Proc. Natl.
Acad. Sci. USA 2008, 105, 6959–6964.
[241] Sambourg, L., Thierry-Mieg, N., New insights into protein-
protein interaction data lead to increased estimates of the
S. cerevisiae interactome size. BMC Bioinform. 2010, 11,
605.
[242] Sowa, M. E., Bennett, E. J., Gygi, S. P., Harper, J. W., Defin-
ing the human deubiquitinating enzyme interaction land-
scape. Cell 2009, 138, 389–403.
[243] Sardiu, M. E., Cai, Y., Jin, J., Swanson, S. K. et al., Prob-
abilistic assembly of human protein interaction networks
from label-free quantitative proteomics. Proc. Natl. Acad.
Sci. USA 2008, 105, 1454–1459.

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1497
[244] Lavallee-Adam, M., Cloutier, P., Coulombe, B., Blanchette,
M., Modeling contaminants in AP-MS/MS experiments.
J. Proteome Res. 2011, 10, 886–895.
[245] Liu, G., Zhang, J., Larsen, B., Stark, C. et al., Prohits: inte-
grated software for mass spectrometry-based interaction
proteomics. Nat. Biotechnol. 2010, 28, 1015–1017.
[246] Choi, H., Larsen, B., Lin, Z.-Y., Breitkreutz, A. et al., Saint:
probabilistic scoring of affinity purification-mass spec-
trometry data. Nat. Methods 2011, 8, U70–U100.
[247] Ranish, J. A., Hahn, S., Lu, Y., Yi, E. C. et al., Identification of
TFB5, a new component of general transcription and DNA
repair factor iih. Nat. Genet. 2004, 36, 707–713.
[248] Trinkle-Mulcahy, L., Andersen, J., Lam, Y. W., Moorhead,
G. et al., Repo-man recruits pp1 gamma to chromatin and
is essential for cell viability. J. Cell Biol. 2006, 172, 679–
692.
[249] Selbach, M., Mann, M., Protein interaction screening by
quantitative immunoprecipitation combined with knock-
down (QUICK). Nat. Methods 2006, 3, 981–983.
[250] Hubner, N. C., Bird, A. W., Cox, J., Splettstoesser, B.
et al., Quantitative proteomics combined with bac trans-
geneomics reveals in vivo protein interactions. J. Cell Biol.
2010, 189, 739–754.
[251] Uhlens, M., Bjorling, E., Agaton, C., Szigyarto, C. A. et al., A
human protein atlas for normal and cancer tissues based
on antibody proteomics. Mol. Cell. Proteomics 2005, 4,
1920–1932.
[252] Persson, A., Hober, S., Uhlen, M., A human protein atlas
based on antibody proteomics. Curr. Opin. Mol. Therap.
2006, 8, 185–190.
[253] Colwill, K., Graslund, S., Renewable prot binder working,
G., A roadmap to generate renewable protein binders
to the human proteome. Nat. Methods 2011, 8, U551–
U552.
[254] Taussig, M. J., Stoevesandt, O., Borrebaeck, C. A. K., Brad-
bury, A. R. et al., Proteomebinders: planning a European
resource of affinity reagents for analysis of the human pro-
teome. Nat. Methods 2007, 4, 13–17.
[255] Malovannaya, A., Lanz, R. B., Jung, S. Y., Bulynko, Y. et al.,
Analysis of the human endogenous coregulator complex-
ome. Cell 2011, 145, 787–799.
[256] Reichert, J. M., Monoclonal antibodies as innovative ther-
apeutics. Curr. Pharm. Biotechnol. 2008, 9, 423–430.
[257] Reichert, J. M., Rosensweig, C. J., Faden, L. B., Dewitz,
M. C., Monoclonal antibody successes in the clinic. Nat.
Biotechnol. 2005, 23, 1073–1078.
[258] Ravelli, R. B. G., Gigant, B., Curmi, P. A., Jourdain, I.
et al., Insight into tubulin regulation from a complex with
colchicine and a stathmin-like domain. Nature 2004, 428,
198–202.
[259] Pommier, Y., Marchand, C., Interfacial inhibitors: targeting
macromolecular complexes. Nat. Rev. Drug Discov. 2012,
11, 25–36.
[260] Wells, J. A., McClendon, C. L., Reaching for high-hanging
fruit in drug discovery at protein-protein interfaces. Nature
2007, 450, 1001–1009.
www.proteomics-journal.com
1498 P. Braun and A.-C. Gingras
[261] Arkin, M. R., Wells, J. A., Small-molecule inhibitors
of protein-protein interactions: progressing towards
the dream. Nat. Rev. Drug Discov. 2004, 3, 301–
317.
[262] Vidal, M., Braun, P., Chen, E., Boeke, J. D. et al., Ge-
netic characterization of a mammalian protein-protein in-
teraction domain by using a yeast reverse two-hybrid
system. Proc. Natl. Acad. Sci. USA 1996, 93, 10321–
10326.
[263] Dreze, M., Charloteaux, B., Milstein, S., Vidalain, P. O. et al.,
‘Edgetic’ perturbation of a C. elegans BCL-2 ortholog. Nat.
Methods 2009, 6, 843–849.
[264] Patel, S., Player, M. R., Small-molecule inhibitors of the
p53-HDM2 interaction for the treatment of cancer. Expert
Opin. Invest. Drug. 2008, 17, 1865–1882.
[265] Barabasi, A. L., Oltvai, Z. N., Network biology: understand-
ing the cell’s functional organization. Nat. Rev. Genet. 2004,
5, 101–113.

C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2012, 12, 1478–1498
[266] Barabasi, A.-L., The network takeover. Nat. Phys. 2012, 8,
14–16.
[267] Chuang, H.-Y., Hofree, M., Ideker, T., A decade of systems
biology. Ann. Rev. Cell Dev. Biol. 2010, 26, 721–744.
[268] Zanzoni, A., Soler-Lopez, M., Aloy, P., A network medicine
approach to human disease. FEBS Lett. 2009, 583, 1759–
1765.
[269] Zhong, Q., Simonis, N., Li, Q. R., Charloteaux, B. et al.,
Edgetic perturbation models of human inherited disorders.
Mol. Syst. Biol. 2009, 5, 321.
[270] Gammie, A. E., Erdeniz, N., Beaver, J., Devlin, B. et al.,
Functional characterization of pathogenic human MSH2
missense mutations in Saccharomyces cerevisiae. Genet-
ics 2007, 177, 707–721.
[271] Yamamoto, Y., Hirai, T., Yamamoto, E., Kawamura, M. et al.,
A rice GID1 suppressor mutant reveals that gibberellin is
not always required for interaction between its receptor,
GID1, and DELLA proteins. Plant Cell 2010, 22, 3589–3602.
www.proteomics-journal.com