Báo cáo khoa học: Sherlock Holmes and the proteome ) a detective story Pier Giorgio Righetti1 and Egisto Boschetti2 potx

REVIEW ARTICLE
Sherlock Holmes and the proteome ) a detective story
Pier Giorgio Righetti
1
and Egisto Boschetti
2
1 Department of Chemistry Materials and Chemical Engineering ‘Giulio Natta’, Polytechnic of Milano, Milan, Italy
2 Ciphergen Biosystems, Fremont, CA, USA
Introduction
The word ‘detective’ originates from the Latin ‘detego’
(detexi, detectum, detegere), i.e. to find out, to discover
(in fact, to remove the teges or tegmen, in English
slang the cover, therefore to uncover!). Modern prote-
ome analysis is a very complex ‘detective story’, which
might baffle even the most famous investigator, Sher-
lock Holmes [1]. The reason is that, in any proteome,
a few proteins dominate the landscape and often oblit-
erate the signal of the rare ones, so that, when the
police reach the scene of the crime, the thin thread of
evidence remains hidden. In addition, proteomes of
any origin can be extremely complex, impervious to
even the most sophisticated analytical tools. For
instance, according to Anderson et al. [2,3], the human
plasma should contain most, if not all, human pro-
teins, as well as proteins derived from viruses, bacteria
and fungi. Also, numerous post-translationally modi-
fied forms of each protein are present, along with,
possibly, millions of distinct clonal immunoglobulin
sequences. To this intrinsic complexity, one can add
the enormous dynamic range, encompassing some 10
orders of magnitude between the least abundant (e.g.

interleukins, at concentrations of < 1 ngÆmL
)1
) and
the most abundant (e.g. albumin,  50 mgÆmL
)1
). For
these reasons, any scientist working on any proteomic
project deserves the title ‘detective’, be it the most
famous Sherlock Holmes, the illustrious Hercule Poirot
[4], or even the clumsy inspecteur Jacques Clouseau,
de la Su
ˆ
rete
´
de Paris [5].
Dozens of published papers have highlighted major
limitations of available technologies for proteome
investigation. Current approaches are incapable of
attaining a complete picture of the proteome, even lim-
ited with respect to structural aspects. For instance,
Keywords
E. coli proteome; ligand library; peptide
ligands; rare proteome; S. cerevisiae
proteome; urine and serum analysis
Correspondence
P. G. Righetti, Department of Chemistry
Materials and Chemical Engineering ‘Giulio
Natta’, Polytechnic of Milano, Via Mancinelli
7, Milano 20133, Italy
Fax: +39 022399 3080

Tel: +39 022399 3016
E-mail:
Note
This lecture was delivered at the 7th Siena
Meeting ‘From Genome to Proteome: Back
to the Future’, September 3–7, 2006, Siena,
Italy.
(Received 5 October, revised 26 November
2006, accepted 13 December 2006)
doi:10.1111/j.1742-4658.2007.05648.x
The performance of a hexapeptide ligand library in capturing the ‘hidden
proteome’ is illustrated and evaluated. This library, insolubilized on an
organic polymer and available under the trade name ‘Equalizer Bead Tech-
nology’, acts by capturing all components of a given proteome, by concen-
trating rare and very rare proteins, and simultaneously diluting the
abundant ones. This results in a proteome of ‘normalized’ relative abun-
dances, amenable to analysis by MS and any other analytical tool. Exam-
ples are given of analysis of human urine and serum, as well as cell and
tissue lysates, such as Escherichia coli and Saccharomyces cerevisiae
extracts. Another important application is impurity tracking and polishing
of recombinant DNA products, especially biopharmaceuticals meant for
human consumption.
FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS 897
strongly alkaline proteins are poorly represented
on classical two-dimensional electrophoresis [6], and
highly hydrophobic proteins cannot be properly solubi-
lized and consequently not analyzed and ⁄ or identified
at all. Electrophoresis-based methods on their own
(still the most commonly used to date) are neither
appropriate for polypeptides of mass lower than

5000 Da nor effective for very alkaline proteins. Only
MS contributes significantly to the analysis of small
polypeptides. To this list of limitations can be added
the fact that post-translational modifications, especially
glycosylations, are still part of the unresolved dilem-
mas. It is estimated that only 20–30% of expressed
proteins are detectable by standard methods to date.
Prefractionation of all possible variants has been
deemed the logical way to go in the attempt to move
in the right direction. As stated by Pedersen et al. [7],
prefractionation could be a formidable tool for ‘mining
below the tip of the iceberg to find low abundance and
membrane proteins’. A wide variety of prefractionation
protocols, exploiting all possible variations of chroma-
tographic and electrophoretic procedures, have been
described (for reviews, see [8–11]). It should be remem-
bered that one of the oldest and still most valid meth-
ods for simplifying a cell proteome is the separation of
cell substructures by the centrifugal cell-fractionation
scheme. This method is well-ingrained in classical bio-
chemical analysis, as developed in the late fifties and
early sixties by de Duve and other research groups. By
this procedure, via a series of runs at different centrifu-
gal forces, one can isolate, in a reasonably pure form,
subcellular organelles, such as nuclei, mitochondria,
lysosomes, peroxisomes, synaptosomes, microbodies
and the like [12].
It should be appreciated that, in the armamentarium
of prefractionation tools available for such complex
analysis, no single method has been sufficient to carry

out this task. The approach that is gaining momentum,
especially in analysis of biological fluids, such as
plasma, sera, cerebrospinal fluid, urine, is sequential or
simultaneous immunoaffinity depletion of the most
abundant proteins present in the samples [13]. How-
ever, even this approach may not be good enough to
gain access to the ‘deep proteome’. Although depletion
of the nine most abundant proteins represents the
removal of as much as 90% of the overall protein
content, the vast number of serum proteins that
comprise the remaining 10% remain dilute, and the
improvement in detecting rare proteins might be quite
disappointing. In fact, Echan et al. [14], using a
commercial column for removal of the top six most
abundant proteins, reported: ‘many of the moderate
and low-intensity protein spots that were detected on
the depleted sample gels were actually detectable on
the unfractionated sample gel’. Another major draw-
back of such immuno-subtraction methods appears to
be co-depletion. As reported by Shen et al. [15], during
depletion of human serum albumin, another 815
species (not including this protein) were co-depleted.
When capturing IgGs, another 2091 species (not inclu-
ding IgG) were co-depleted, among which 56% were
antibody sequences and the other 44% included
low-abundance cytokines and related proteins. Para-
doxically, in the sera thus subtracted from just these
two major proteins, only 1391 free proteins could be
detected. Ironically, most of the newly discovered spe-
cies were found in the two fractions that had to be dis-

carded, not in the fraction meant to be recovered.
Aware of all the drawbacks discussed above, we
recently proposed a novel method for capturing and
identifying the ‘hidden proteome’, called Protein
Equalizer Technology. It consists of a solid-phase com-
binatorial library of hexapeptides, which are coupled,
via a short spacer, on poly(hydroxymethacrylate)
beads, by a modified Merrifield approach [16]. The
properties of these beads and their application to a
variety of proteomic analyses are reported.
The Equalizer Bead technology
Equalizer Beads comprise a solid-phase combinatorial
library of hexapeptides that are synthesized via a short
spacer on a poly(hydroxymethacrylate) substrate,
according to a modified Merrifield approach, by using
the split, couple, recombine method. Briefly, a batch of
millions of microscopic porous chromatographic beads
is divided up into several equal reaction vessels. The
number of reaction vessels is the same as the number
of building blocks (e.g. amino acids) used for the pro-
duction of the ligand sequences. Each bead vessel
receives a different building block, which is chemically
attached to the beads. The different bead vessels are
then mixed together, extensively washed, chemical pro-
tection groups are removed, and finally the batch is
split up again into the same number of sub-batches as
before. The process of building block coupling at the
extremity of the first attached chemical group is then
started again. Thus a second building block is attached
in a combinatorial manner. The process is repeated

until a sequence of the desired length is produced (this
process is detailed in Lam et al. [16]).
The ligands are represented throughout the beads’
porous structure and can achieve an amount of
 15 pmol per bead of the same hexapeptide distri-
buted throughout the core of the pearl. This amounts
to a ligand density of  40–60 lmolÆ mL
)1
bead volume
Sherlock Holmes and the proteome P. G. Righetti and E. Boschetti
898 FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS
(average bead diameter  60 lm). As a result of the
nonrandomized combinatorial hexapeptide construc-
tion, each bead has many copies of a single, unique
ligand, and each bead has a different ligand from every
other bead. Considering that, for the synthesis of a
protein, many amino acids are used, the resulting
library contains a population of linear hexapeptides
amounting to millions of different ligands. Such a vast
population of baits means that, in principle, every
protein present in a complex proteome (be it a bio-
logical fluid or a tissue or cell lysate of any origin)
potentially has a bead partner carrying the peptide
ligand with which it is able to interact under the
well-known affinity chromatography mechanism. As
demonstrated in another article [17], each bead cap-
tures a different dominant protein and co-adsorbs a
small amount of a very few other species. The principle
has been used to identify the hexapeptide ligand struc-
ture specific to selected proteins [18,19]. It should be

noted, however, that a given protein can adsorb to
more than one peptide ligand structure. The latter
governs the affinity constant value and can be used as
the basis for selecting the interacting ligand for affinity
chromatography purification (see papers referenced
above). When proteins have multiple possibilities for
peptide–ligand interaction, they are more enriched
than others: this is clearly the case for apolipoproteins
from human serum, for example. Lengthening the bait
to a heptamer or even an octamer would generate a
much larger number of diverse ligands, probably con-
siderably more heterogeneous than all the diverse pro-
teins synthesized by all known living organisms. The
incorporation of d-enantiomers and even unnatural
amino acids into linear, branched, or circular peptides
would generate a potential library diversity that would
be practically unlimited and would surely contain a lig-
and to every protein present in a biological sample.
The use of a hexapeptide ligand to establish an affinity
interaction might be considered a rather weak binding
event; however, experience has shown that, in fact,
such a complex can have very high affinity and require
very strong elution conditions. The hexameric ligands
are linked to the organic polymer in such a way as to
be stable under typical experimental conditions, such
as prolonged incubation at reduced or elevated pH
and ionic strengths and organic solvents used to elicit
complex formation with cell ⁄ tissue lysates and subse-
quent elution from the beads. The initial article outlin-
ing the synthesis of the beads and some of their

fundamental properties has recently been published
[20], together with reviews describing the basic con-
cepts [9,21,22]. The mechanism of action of the Equal-
izer Beads is illustrated in Fig. 1. Rather than acting in
depletion methods, or by selecting a given population
of species, via any possible prefractionation tool, the
beads are meant to adsorb just about any component
of the proteome under analysis, but in a very unusual
way. As shown in the lower left graph (Fig. 1), the
relative abundance of proteins is such that a few are
present in a large excess, whereas the vast majority are
present at a concentration often considerably below
the detection limit. As, in principle, each protein
species has the same number of baits available on the
adsorbing pearls, the species present in vast excess
quickly saturate their ligand, leaving the remainder
unbound in solution. In contrast, rare and very rare
species keep being adsorbed to their respective ligand,
thus being depleted (or very nearly so) from the
Fig. 1. Illustration of the mechanism of action of Equalizer Beads. Bottom panel: relative protein abundances in a generic proteome (left) ver-
sus ‘normalized’ protein abundances after treatment with the hexapeptide ligand library (right). Upper right: adsorbed proteins can be eluted
en bloc, or with sequential treatments of increasing strength.
P. G. Righetti and E. Boschetti Sherlock Holmes and the proteome
FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS 899
solution. This results in ‘normalization’ of the relative
abundance ratios (lower right panel, Fig. 1), rendering
the vast majority of proteins amenable to further ana-
lysis and identification by MS or any other appropriate
tool.
The technique described is not yet commercially

available; however, using the detailed description in
previous papers [20–22], it could be implemented by
using any peptide libraries made on solid phases.
Analysis of biological fluids
We will give some examples of biological extracts that
have been analysed with the help of the Equalizer
Bead technique. For decades, clinical chemistry
research has focused on finding, in any tissue speci-
men, but especially body fluids (plasma, urine, tears,
lymph, seminal plasma, milk, saliva, spinal fluid), new
indicators of disease. The search for biomarkers in
body fluids is particularly attractive, as their collection
is minimally invasive or, in the case of urine, not inva-
sive at all. However, even body fluids are not free from
the problems that have so far hampered the discovery
of novel markers; for example, both plasma and serum
exhibit tremendous variations in individual protein
abundance, typically of the order of 10
10
or more, with
the result that, in any typical two-dimensional map,
only the high-abundance proteins are revealed. In the
case of urine, the problems are further aggravated by
the very low protein content, requiring a concentration
step of 100–1000-fold, coupled with its high level of
salts, which need to be removed before any analytical
step. When urine from healthy donors was treated with
Equalizer Bead technology and the eluate analysed by
MS, the results were quite impressive. Control urine
samples revealed a total of 96 unique gene products.

In contrast, the first eluate (in 2.2 m thiourea, 7 m urea
and 4% CHAPS) allowed identification of 334 unique
protein species, and the second eluate (in 9 m urea
titrated to pH 3.8 with 5% acetic acid) an additional
148 species. By eliminating the redundancies and
counting all the species detected, we arrived at a total
of 471 unique protein species in urine [23]. This com-
pares quite favourably with the best data available in
the literature so far, which were obtained using much
more complex technologies and experimental proto-
cols, such as the data of Pieper et al. [24], who repor-
ted 150 unique protein annotations (obtained by
extensive sample prefractionation and two-dimensional
map analysis). However, in the most recent report [25],
1543 proteins were identified in urine samples obtained
from 10 healthy donors, using highly sophisticated
methodology involving analysis of the tryptic digests
via a linear ion trap-Fourier transform (LTQ-FT) and
a linear ion trap-orbitrap (LTQ-Orbitrap) mass spec-
trometers.
A similar approach was adopted, exploiting our pep-
tide library beads, for a large-scale proteomic study of
human blood serum. After ‘equalizing’ sera on the
hexameric peptide baits, analysis by liquid chromato-
graphy of trypsin hydrolyzates coupled with high-
resolution MS resulted in the identification of 3869 or
1559 proteins, depending on how the 95% confidence
was estimated. In either case, the analysis showed that
ligand beads were able to capture a large number of
proteins in a single operation [26]. To determine what

fraction of our 1559 protein dataset represents novel
serum proteins, we compared our protein list with
other published, large-scale human serum datasets. We
chose the results of a study coordinated by the HUPO
Plasma Protein Project (HPPP) [27]. This study reports
a total of 889 unique gene products. Thus, it can be
seen that this novel technique offers some unique
advantages over standard methodologies, even when
data are pooled from a large number of laboratories.
Of the proteins identified here, 86% had not previously
been reported in the HUPO-coordinated effort of 35
laboratories quoted here. As a visual example, we
report here one-dimensional SDS ⁄ PAGE profiles of
human (Fig. 2) and mouse (Fig. 3) sera, before and
after treatment with Equalizer Beads. In the first case,
the proteins were eluted en masse from the beads with
6 m guanidine hydrochloride, whereas in the second
case, the adsorbed species were sequentially eluted first
with 1 m NaCl pH 7.0, followed by 3 m guanidine
hydrochloride, pH 6.0, and finally by 9 m urea ⁄ citrate,
pH 3.8, each treatment being able to interfere with dif-
ferent types of interaction among the proteins and the
baits. In both cases, the dramatic increase in the num-
ber of protein zones throughout the mass range (from
5 to > 200 kDa) can be seen at a glance. In the search
for novel biomarkers, Equalizer Beads have also been
applied to plasma and sera by Lathrop et al. [28].
Analysis of cells and tissues
Although biological fluids appear to be the ideal sub-
strate for Equalizer Bead treatment, the treatment

should also work, in principle, in the case of cell and
tissue extracts from any origin. In fact, cell and tissue
lysates should also exhibit a similar disparity in protein
concentration ranges to that found in body fluids. It is
a fact that, when a total cell extract is examined, for
instance, by two-dimensional maps, the most intense
spots are those from cytoskeletal proteins and house-
keeping proteins. Here also rare and very rare proteins
Sherlock Holmes and the proteome P. G. Righetti and E. Boschetti
900 FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS
cannot be brought to the forefront. As an example,
Fig. 4A,B shows SDS ⁄ PAGE profiles of Escherichia
coli and Saccharomyces cerevisiae extract, respectively,
before and after treatment with Equalizer Beads. In
both cases, it can be appreciated that a much larger
number of bands is visible over the entire trace, inclu-
ding lower molecular mass protein ⁄ peptides that often
escape detection by conventional means. In the partic-
ular case of E. coli, bands were cut out from the elec-
trophoresis gel (Fig. 4A, lane b) to identify proteins by
in-gel digestion followed by liquid chromatography-
MS ⁄ MS analysis. The protein identity of several of
them was reported by Thulasiraman et al. [20]. All
these proteins were of low abundance. For instance, on
the basis of previous work, ADP-l-glycero-b-manno-
heptose-6-epimerase is present at  220 copies per cell;
another five enzymes listed (NADH–quinone oxidore-
ductase chain C ⁄ D; tagatose-6-phosphate kinase, gat-Z;
glutamate-1-semialdehyde 2,1-aminomutase; glycine ace-
tyltransferase; galactitol-1-phosphate-5-dehydrogenase)

were not previously detected by two-dimensional elec-
trophoretic analysis of the whole lysate because of
their low concentrations; moreover, tagatose-6-phos-
phate kinase gat-Z was previously reported only by
DNA sequence.
Impurity tracking and polishing of
recombinant DNA biotech products
Another important field of application of the Equalizer
Bead method is capturing and ‘amplifying’ impurities
present at trace levels in recombinant DNA products,
especially those meant for human consumption. Most
biopharmaceuticals today are products of recombinant
DNA technology or derived from human plasma.
Recombinant proteins are expressed in selected host
cells under controlled conditions, whereas human
plasma-derived products are extracted from pooled
human plasma. Both are complex starting materials
with thousands of proteins that are potential impurities
of the final product that may, in rare cases, cause
adverse events in the patient ranging from a slight
fever to long-term immunogenicity to toxic and even,
in rare cases, fatal events. Host cells used for the bio-
synthesis of recombinant proteins are relatively com-
plex systems extending from bacteria (e.g. E. coli), to
yeasts (e.g. Pichia pastoris) to eukaryotic cells, such as
188 kDa
98
62
49
38

28
17
14
M.wt Stds
Pooled Elution
3
rd
wash
2
nd
wash
2
nd
wash
1
st
wash
1sth elution
FT
Serum
Fig. 2. Analysis of human serum proteins before and after Equalizer
Bead treatment. One-dimensional SDS ⁄ PAGE profiles. Staining
with colloidal Coomassie Blue. Lanes 1)4 refer to control serum
(untreated), flow through (FT) after bead treatment, followed by
two washing steps, respectively. Lanes 5)8 refer to first elution
en block (with 6
M guanidine hydrochloride, pH 6.0) followed by
two washing steps, and finally SDS ⁄ PAGE of all pooled eluates,
respectively. Lane 9: SDS profile of molecular mass standards.
Fig. 3. Analysis of mouse serum proteins by SDS ⁄ PAGE. Lanes: 1,

molecular mass ladder; 2, control (untreated) mouse serum; 3, 1
M
NaCl, pH 7.0, eluate from Equalizer Beads; 4, 3 M guanidine hydro-
chloride, pH 6.0, eluate; 5, 9
M urea in citrate, pH 3.8 eluate. Stain-
ing with colloidal Coomassie Blue.
P. G. Righetti and E. Boschetti Sherlock Holmes and the proteome
FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS 901
Chinese hamster ovary (CHO) cells. During culture,
these cells secrete a very large number of their own
proteins, which can easily contaminate the recombin-
ant DNA product. Even after sophisticated purifica-
tion steps, significant levels of host cell proteins may
remain in the final purified biopharmaceutical.
Although host cell impurities are mostly innocuous to
the patient, regulatory agencies require demonstration
that host cell proteins are not only minimized but also
analyzed with the most sensitive available methods.
Current analytical methods are limited in number and
also not sufficiently sensitive for the detection of trace
levels of host cell proteins. Current HPLC techniques
have good resolution; however, they suffer from low
sensitivity, the possibility of nonspecific binding, and
subjective interpretation. Electrophoretic analytical
methods (e.g. SDS ⁄ PAGE with silver staining) also
offer good resolution, but sensitivity is low and the
interpretation is also very subjective. Immunological
determination is more specific than electrophoretic
techniques and chromatography; however, analytical
results depend on variation in the affinity constants

of the selected antibodies. In conclusion, all detection
methods for host cell proteins have a challenging
problem, namely, how to deal with very low concen-
trations of contaminating proteins present in ‘pure’
biopharmaceuticals after separation ⁄ purification with
current processing techniques.
Aware of these limitations, we have used the Equal-
izer Bead library to track these very low level impurit-
ies, and have already reported a couple of most
promising applications [29,30]. We give here an exam-
ple of such an impurity ‘amplification’, as applied to
purified monoclonal antibodies produced in hybridoma
cells. Figure 5A shows a two-dimensional map of
control monoclonal antibodies, purified with a merca-
pto-ethyl-pyridine resin [31,32], where very few con-
taminants are visible. After treatment with Equalizer
Beads (Fig. 5B), a large number of new spots appear.
Most of them were excised, digested and subjected to
liquid chromatography-MS ⁄ MS analysis. Two classes
of ‘contaminants’ could be detected: (a) mouse hybri-
doma proteins and culture broth proteins (notably
BSA along with its fragments); (b) a large number of
fragments of the monoclonal antibodies produced.
This seems to be a general trend with all recombinant
DNA products we have analysed so far. It should be
emphasized here that the unique ability of Equalizer
Beads to track and concentrate such impurities is a
process that could be (the necessary changes having
been made) compared with PCR for amplification of
nucleic acid fragments, allowing the detection of pro-

teins that would otherwise be invisible. We have esti-
mated that this amplification-like process can increase
the local concentration of such impurities in the
final product by three to four orders of magnitude
210
105
34
17
7
cba
BA
ba
Fig. 4. Analysis of cell lysates before and
after Equalizer Bead treatment by
SDS ⁄ PAGE. (A) E. coli extract (a, control; b,
Equalizer Bead eluate in 9
M urea and cit-
rate, pH 3.5). (B) S. cerevisiae extract (a,
molecular mass ladder; b, control; c, Equal-
izer Bead eluate in 9
M urea ⁄ citrate, pH 3.5).
Staining with colloidal Coomassie Blue.
Sherlock Holmes and the proteome P. G. Righetti and E. Boschetti
902 FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS
depending on the amount of extract loaded and the
bead volume.
Analysis of purification may also have utility during
the development of second-generation processes for a
given biopharmaceutical, where demonstration of com-
parability is to be made not only for the degree of pur-

ity of the target protein but also for the qualitative
and quantitative presence of traces of impurity.
Equalizer Beads can be applied here for two proces-
ses: in the first instance, for tracking and concentrating
such impurities, so as to render them amenable to
identification by MS and other analytical techniques
(in this case, a small amount of beads is incubated
with large sample volumes and quantities); by the same
token, if now the beads are in excess over the sample
amount, the beads will also remove such impurities
and thus would be the ideal final ‘polishing’ step for
such biopharmaceuticals [29].
A panacea?
It is intrinsic to human nature to try to overemphasize
the importance of any innovation, with claims often
vastly exceeding what can be achieved in practice
with any novel concept or methodology; in daily use,
such innovations rarely meet the expectations. Science
grows by small increments, quantum jumps being rare
events. Panaceas existed only in legends and the dreams
of sorcerers and healers, and they were scorned in the
famous comedy of Molie
`
re, Le Malade Imaginaire,
where the candidate physicians would advocate only a
single remedy for any possible disease, and hardly a
mild one at that (clysterium practicare, postea salassare,
infinem purgare). We will thus briefly highlight the major
advantages as well as the limitations of the present
approach. The advantages are at least twofold: while

this highly diversified ligand library is able to greatly
concentrate rare and very rare proteins, bringing them
to the forefront, it simultaneously dilutes the most
abundant ones, as only a tiny fraction of them is recov-
ered by saturation of their respective ligands. This extra
benefit cannot be overemphasized. For example, anyone
working with sera knows well that albumin obliterates
the signal of most proteins co-focusing in the same pI
region. It just so happens that human serum albumin
focuses in the pH 5–6 region (under denaturing condi-
tions), because of a multitude of isoforms [33]. Thus, all
proteins that focus in this region have to fight against
this ‘Goliath’ for survival. From this point of view, as
Equalizer Beads are not meant to select a single protein,
such as antibodies, or protein family, such as lectins, or
to capture specific components, but rather to embrace
all proteins in a proteome, they are ecumenical (or at
least they try to be), i.e. they accept and adopt all
‘faiths, colours, races and creeds’. In addition, they
introduce ‘democracy’ in a rather ‘oligarchic’ (some
would say ‘plutocratic’) proteome. Another major
bonus of the approach described is the capture and
adsorption of a high proportion of small and large pep-
tides (in the 600–8000-Da range) that are normally lost
upon two-dimensional electrophoretic mapping. Such a
peptide population in human sera may be of particular
importance as it may contain protein cleavage products
of diagnostic value [34].
There is at least one major limitation to the present
method: owing to the fact that the interaction mechan-

ism is rather delicate (it encompasses all types of bonds
250-
150-
100-
75-
50-
37-
25-
20-
15-
10-
M
r
(kDa)
M
r
(kDa)
3 pI 10
A
250-
150-
100-
75-
50-
37-
25-
20-
15-
10-
3 pI 10

B
Fig. 5. Analysis of monoclonal Igs from mouse hybridomas, purified
by mercapto-ethyl-pyridine–HyperCel chromatography, via two-
dimensional maps. (A) Control monoclonal antibodies (untreated);
(B) monoclonal antibodies after Equalizer Bead treatment. Newly
revealed spots eluted and analysed by liquid chromatography-
MS ⁄ MS. Staining with Sypro Ruby. First dimension: nonlinear
pH 3–10 immobilized pH gradients. Second dimension: SDS ⁄ PAGE
in a 8–10% polyacrylamide gel slab.
P. G. Righetti and E. Boschetti Sherlock Holmes and the proteome
FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS 903
that help to stabilize the tridimensional structure of
proteins, such as ionic, hydrogen bonding and hydro-
phobic association, and other weak interactions such
as van der Waals forces) adsorption can only be
obtained under native physiological conditions, i.e. in
the absence of strong denaturants. Thus, membrane
and very hydrophobic proteins, which normally require
a strong solubilizing agent for dissolution, cannot be
recovered, as mild conditions are required when Equal-
izer Beads are incubated with any proteome; for exam-
ple, TUC (thiourea, urea and CHAPS solubilizing
solution), a classical solubilizing cocktail in two-dimen-
sional maps, is typically used for desorption of pro-
teins bound to the beads.
Another matter of concern regards the possibility of
abnormal binding of proteins to the beads, leading to
unequal situations. It is unrealistic to think that all
proteins will behave well towards the adsorbing ligand
library. Working with sera, we have found at least one

protein with unexpected behaviour: apolipoprotein J
(Apo J), which is greatly enriched compared with all
other serum components, rendering it the most abun-
dant component after equalization. Apo J possesses a
large number of binding sites for several components,
suggesting that it may recognize more than one hexa-
peptide ligand, thus saturating an abnormal number of
sites in a larger bead population compared with other
‘well-behaved’ proteins. Close examination of two-
dimensional maps suggests that a few other proteins
might exhibit similar behaviour, although to what
extent this abnormal behaviour will affect the total
proteome of a tissue is yet to be investigated.
Conclusions
We briefly summarize here the major points worth
considering when using the heaxapeptide combinatorial
library in any proteome analysis. Here is what can be
accomplished with this method: (a) amplification,
detection and identification of protein traces, partic-
ularly in various biological fluids and extracts, detec-
tion of host cell protein in recombinant pure proteins;
(b) identification of specific ligands for protein; (c) pol-
ishing step in downstream processing; (d) discovery of
biomarkers of diagnostic interest; (e) protein–protein
interaction studies.
Acknowledgements
PGR is supported by grants from the European Com-
munity (Allergy card), by PRIN 2006 (MIUR, Rome)
and by Fondazione Cariplo. We thank providers of
biological fluids, such as E. coli extracts (Dr S. Lin)

and S. cerevisiae extract (Dr M. Toledano and Dr N.
le Moan, CEA Saclay, France), as well as providers of
experimental data (Dr V. Thulasiraman, Dr L. Guer-
rier, Dr F. Fortis, Dr P. Antonioli).
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