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: 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. 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). References 1 Conan Doyle A. (2005) The Casebook of Sherlock Hol- mes. BBC Consumer Publishing, London. 2 Anderson NL & Anderson NG (2002) The human plasma proteome: history, character and diagnostic pro- spects. Mol Cell Proteomics 1, 845–867. 3 Anderson LN, Polanski M, Pieper R, Gatlin T, Tiruma- lai RS, Conrads TP, Veenstra TD, Adkins JN, Pounds JG, Fagan R, et al. (2004) The human plasma proteome. Mol Cell Proteomics 3, 311–326. 4 Christie A (1934) Murder on the Orient Express. Arche- tte, Paris. 5 Edwards B (1963) The Pink Panther. Paramount, Hollywood, CA. 6 Bae SH, Harris AG, Hains PG, Chen H, Garfin DE, Hazell SL, Paik YK, Walsh B & Cordwell SJ (2003) Strategies for the enrichment and identification of basic proteins in proteome projects. Proteomics 3, 569–579. 7 Pedersen SK, Harry JL, Sebastian L, Baker J, Traini MD, McCarthy JT, Manoharan A, Wilkins MR, Goo- ley AA, et al. (2003) Unseen proteome: mining below the tip of the iceberg to find low abundance and mem- brane proteins. J Proteome Res 2, 303–312. 8 Righetti PG, Castagna A, Herbert B, Reymond F & Rossier JS (2003) Prefractionation techniques in pro- teome analysis. Proteomics 3 , 1397–1407. 9 Righetti PG, Castagna A, Antonioli P & Boschetti E (2005) Prefractionation techniques in proteome analysis: the mining tools of the third millennium. Electrophoresis 26, 297–319. 10 Garbis S, Lubec G & Fountoulakis M (2005) Limita- tions of current proteomics technologies. J Chromatogr A 1077, 1–18. 11 Vlahou A & Fountoulakis M (2005) Proteomic appro- aches in the search for disease biomarkers. J Chromatogr B 814 , 11–19. 12 Huber LA, Pfaller K & Vietor I (2003) Organelle pro- teomics: implications for subcellular fractionation in proteomics. Circ Res 92, 962–968. 13 Pieper R, Gatlin CL, Makusky AJ, Russo PS, Schatz CR, Miller SS, Su Q, McGrath AM, Estock MA, Parmar PP, et al. (2003) The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3, 1345–1364. 14 Echan LA, Tang HY, Ali-Khan N, Lee KB & Speicher DW (2005) Depletion of multiple high-abundance Sherlock Holmes and the proteome P. G. Righetti and E. Boschetti 904 FEBS Journal 274 (2007) 897–905 ª 2007 The Authors Journal compilation ª 2007 FEBS proteins improves the protein profiling capacities of human serum and plasma. Proteomics 5, 3292–3303. 15 Shen Y, Kim J, Strittmatter E, : F, Jacobs JM, CampDG, IIFang R, Tolie ´ N, Moore RJ & Smith RD (2005) Characterization of the human blood plasma proteome. Proteomics 5, 4034–4045. 16 Lam KS, Salmon SE, Hersh EM, Hruby VJ, Kazmier- ski WM & Knapp RJ (1991) A new type of synthetic peptide library for identifying ligand-binding activity. Nature 354, 82–84. 17 Boschetti E, Lomas L & Righetti PG (2007) Romancing the ‘hidden proteome’, Anno Domini two zero zero seven. J Chromatogr A in press. 18 Buettner JA, Dadd CA, Baumbach GA, Masecar BL & Hammond DJ (1966) Chemically derived peptides libraries: a new resin and methodology for lead identifi- cation. Int J Pept Protein Res 47, 70–83. 19 Kaufman DB, Hentsch M, Baumbach GA, Buettner JA, Dadd CA, Huang PY, Hammond DJ & Carbonnel RG (2002) Affinity purification of fibrinogen using a ligand from a peptide library. Biotechnol Bioeng 77, 278–289. 20 Thulasiraman V, Lin S, Gheorghiu L, Lathrop J, Lomas L, Hammond D & Boschetti E (2005) Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands. Electro- phoresis 26, 3561–3571. 21 Righetti PG, Castagna A, Antonucci F, Piubelli C, Cecconi D, Campostrini N, Rustichelli C, Antonioli P, Zanusso G, Monaco S, et al. (2005) Proteome analysis in the clinical chemistry laboratory: myth or reality? Clin Chim Acta 357, 123–139. 22 Righetti PG, Boschetti E, Lomas L & Citterio A (2006) Protein Equalizer technology: the quest for a ‘democra- tic proteome’. Proteomics 6, 3980–3992. 23 Castagna A, Cecconi D, Sennels L, Rappsilber J, Guer- rier L, Fortis F, Boschetti E, Lomas L & Righetti PG (2005) Exploring the hidden human urinary proteome via ligand library beads. J Proteome Res 4, 1917–1930. 24 Pieper R, Gatlin CL, McGrath AM, Makusky AJ, Mondal M, Seonarain M, Field E, Schatz CR, Estock MA, Ahmed N, et al. (2004) Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electro- phoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 4, 1159–1174. 25 Adachi J, Kumar C, Zhang Y, Olsen JV & Mann M (2006) The human urinary proteome contains more than 1500 proteins including a large proportion of mem- branes proteins. Genome Biol 7, R80–R90. 26 Sennels L, Salek M, Lomas L, Boschetti E, Righetti PG & Rappsilber J (2007) Proteomic analysis of human blood serum using peptide library beads. Mol Cell Proteomics in press. 27 States DJ, Omenn GS, Blackwell TW, Fermin D, Eng J, Speicher DW & Hanash SM (2006) Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collabora- tive study. Nat Biotechnol 24, 333–338. 28 Lathrop JT, Hayes TK, Carrick K & Hammond DJ (2005) Rarity gives a charm: evaluation of trace proteins in plasma and serum. Expert Rev Proteomics 2, 393– 406. 29 Fortis F, Guerrier L, Righetti PG, Antonioli P & Boschetti E (2006) A new approach for the removal of protein impurities from purified biologicals using combi- natorial solid-phase ligand libraries. Electrophoresis 27, 3018–3027. 30 Fre ´ deric Fortis F, Guerrier L, Areces LB, Antonioli P, Hayes T, Carrick K, Hammond D, Boschetti E & Righ- etti PG (2006) A new approach for the detection and identification of protein impurities using combinatorial solid-phase ligand libraries. J Proteome Res 5, 2577– 2585. 31 Luc Guerrier Girot P, Schwartz W & Boschetti E (2000) A new method for the selective capture of antibodies under physiological conditions. Bioseparation 9, 211–221. 32 Warren Schwartz Judd D, Wysocki M, Guerrier L, Birck-Wilson E & Boschetti E (2001) Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies. J Chromatogr 908, 251–263. 33 Williamson AR, Salaman MR & : Kreth HW (1973) Microheterogeneity and allomorphism of proteins. Ann N Y Acad Sci 209, 210–2245. 34 Liotta LA & Petricoin EF (2006) Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold. J Clin Invest 116, 26–30. 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 905 . ‘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. REVIEW ARTICLE Sherlock Holmes and the proteome ) a detective story Pier Giorgio Righetti 1 and Egisto Boschetti 2 1 Department of Chemistry Materials and