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
Trang 1Sherlock Holmes and the proteome ) a detective story
Pier Giorgio Righetti1 and Egisto Boschetti2
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 Suˆrete´ 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: piergiorgio.righetti@polimi.it
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
Trang 2strongly 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 lmolÆmL)1bead volume
Trang 3(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.
Trang 4solution 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 1010or 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
Trang 5cannot 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
rd wash
st wash
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.
Trang 6Chinese 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
c b
a
B A
b
a
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.
Trang 7depending 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-Mr
(kDa)
Mr
(kDa)
A
250-
150-
100-
75-
50-
37-
25-
20-
15-
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.
Trang 8that 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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