Bioscience Reports, Vol. 25, Nos. 1/2, February/April 2005 ( 2005) DOI: 10.1007/s10540-005-2844-2 How to Bring the ‘‘Unseen’’ Proteome to the Limelight via Electrophoretic Pre-Fractionation Techniques Pier Giorgio Righetti,1,4 Annalisa Castagna,1 Ben Herbert,2 and Giovanni Candiano3 The present review reports a panoply of electrophoretic methods as pre-fractionation tools in proteomic investigations in preparation for mass spectrometry or two-dimensional electrophoresis map analysis. Such electrophoretic pre-fractionation protocols include all those electrokinetic methodologies which are performed in free solution, most of them relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separations are multi-chamber apparatuses, such as the multi-compartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads, Oﬀ-Gel electrophoresis in a multi-cup device and the Rotofor, an instrument also based on a multi-chamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other free-ﬂow systems, as well as miniaturized chambers, are also described. KEY WORDS: Proteomics; pre-fractionation; isoelectric focusing; two-dimensional maps. ABBREVIATIONS: FFE: free-ﬂow electrophoresis; FF-IEF: free-ﬂow isoelectric focusing; IPG: immobilized pH gradients; CBI: codon bias index; EOF: electroendoosmotic ﬂow; 2-DE: two-dimensional electrophoresis. INTRODUCTION Although, at the latest count, the total number of coding genes in humans would appear to oscillate between only 25,000 and 30,000 (Southan, 2004), the complexity of the human proteome could, nevertheless, be overwhelming. An example on such a vast complexity and thus on the stringent need for pre-fractionation in proteome analysis comes from some recent articles on the plasma proteome (Anderson and Anderson, 2002; Pieper et al., 2003). If one assumes that there are just 500 true ‘‘plasma proteins’’, each present in 20 variously glycosylated forms and in ﬁve different sizes, one would end up with 50,000 molecular forms. If one further hypothesizes that the ca. 30,000 gene products in the human proteome exist on average as 10 splice variants, cleavage products and post-translational modiﬁcations, 1 Department of Industrial and Agricultural Biotechnologies, University of Verona, Strada Le Grazie 15, 37134, Verona, Italy. 2 Proteome Systems, 35 Waterloo Rd, North Ryde, 2113, Sydney, NSW, Australia. 3 Laboratory of Physiopathology of Uremia, G. Gaslini Children’s Hospital, 16148, Genova, Italy. 4 To whom should be addressed. E-mail: [email protected] 3 0144-8463/05/0400-0003/0 2005 Springer Science+Business Media, Inc. 4 Righetti, Castagna, Herbert, and Candiano this would yield some additional 300,000 protein forms. Rammensee (2004) reported that, by splicing proteins into small pieces, stitching diﬀerent portions together, and then cutting out amino acids sequences from the melded pieces, cells can manufacture a very large variety of new forms from the original gene coded proteins. Moreover single proteins such as antibodies might contain more than 1,000,000 diﬀerent epitopes sequences which add to the complexity of the serum content. To this very intricate situation, additional diﬃculties come from the fact that the dynamic range, at least in serum, might be more than 10 orders of magnitude. In essence it is clear that a natural way to analyze the content of a proteome or even checking phenotyping differences, is to pre-fractionate the proteome into discrete groups and then analyze separately each group. Some recent papers suggest that a problem of this vast complexity could be overcome not by pre-fractionation but rather by running a series of narrow-range IPG strips (covering no more than 1 pH unit). Hoving et al. (2000) and Westbrook et al. (2001) note that ‘‘zoom’’ and ‘‘ultra-zoom’’ gels are quite important for avoiding or at least minimizing the problem of spot overlapping in 2-DE, an ever present hazard in 2-DE maps (see Pietrogrande et al., 2002, 2003; Campostrini et al., 2005). The use of large-size gel slabs (18-cm or longer in the ﬁrst dimension, 18 · 20 cm, or larger, in the second dimension), would even dramatically increase the resolution, as reported by Corthals et al. (2000) and Wildgruber et al. (2000). By that way it seems possible to make portions of bi-dimensional mappings without the necessity of pre-fractionating the sample. The entire, wide-range map would then be electronically reconstructed by stitching together the narrow-range maps. In practice, however, it remains the fact that, even when using very narrow IPG strips, they have to be loaded with the entire tissue lysate with consequent massive precipitation, along with the additional drawback that the proteins which should focus in the chosen narrow-range IPG interval will be strongly under-represented because they will be only a small fraction of the entire sample loaded (Herbert et al., 2004). These serious issues have been debated in a recent work by Gygi et al. (2000), who argued that, in 2D gels, proteins from genes with codon bias values of <0.1 (low-abundance species), large-size proteins (>100 kDa) and most membrane proteins could not be found. Several published papers highlight major limitations of available technologies for proteome investigations. Current approaches are qualiﬁed as incapable of having a whole vision of the proteome, even limited to structural aspects. For instance strongly alkaline proteins are poorly represented when using classical two-dimensional electrophoresis, as underlined by Bae et al. (2003), and highly hydrophobic proteins cannot be properly solubilized and consequently not analyzed and/or identiﬁed. Electrophoresis-based methods taken alone (still the most commonly used to date) are neither appropriate for polypeptides of masses lower than 5000 Da, nor eﬀective for very alkaline proteins. Only mass spectrometry contributes signiﬁcantly to the analysis of low sized polypeptides. To this panel it is to be added that posttranslational modiﬁcations and especially glycosylations are still part of the nonresolved dilemmas. In this situation authors estimate that only about 20 30% of expressed proteins are detectable by standard methods to date. Pre-fractionation, in all of its possible variants, as here reviewed, appears to be the logical way to follow in the attempt to make a step in the right direction. As elegantly stated by Pedersen et al. (2003), in fact, pre-fractionation could be a How to Bring the ‘‘Unseen’’ Proteome to the Limelight 5 formidable tool for ‘‘mining below the tip of the iceberg to ﬁnd low abundance and membrane proteins’’. So, let us wear our proper mining tools, a pickaxe, acetylene lamp, helmet and goggles and descend deep inside the mine to start our digging. Although several types of pre-fractionation tools exist, we will limit this review only to electrophoretic techniques. Centrifugal pre-fractionation, for isolation of cell organelles, has been surveyed in a number of reports (Cordwell et al., 2000; Corthals et al., 2000; Jung et al., 2000; Dreger, 2003; Huber et al., 2003; Stannard et al., 2004). Chromatographic techniques have also been covered by several authors including us (Lopez, 2000; Isaaq et al., 2002; Righetti et al., 2003a, 2003b; Lescuyer et al., 2004). PREPARATIVE ZONE ELECTROPHORESIS Although plain zone electrophoresis in gel cylinders would appear to be just about the last resort, it was recently applied by Fountoulakis and Juranville (2003) to the enrichment of low-abundance brain proteins, by eluting some 80 fractions (each of 10 ml) from a 11%T gel equilibrated with 0.1% lithium dodecyl sulphate (LDS). Interestingly, they could enrich relatively low molecular mass proteins, such as hippocalcin, visinin, 14-3-3 proteins. By the same token, one could perhaps exploit the electrosmotic-pump-driven apparatus of Hayakawa et al. (2003), although gelbased systems do not appear to be popular in pre-fractionation protocols. CONTINUOUS ELECTROPHORESIS IN FREE LIQUID FILMS Over gel phases, this technique has the advantage that much higher sample loads can be applied, coupled to the absence of possible protein modiﬁcations induced by free monomers always present in gel phases (Chiari et al., 1992; Bordini et al., 2000). Present equipment derives from the concepts and instrumentation of Hannig (1967, 1982), by which the electrolyte solution ﬂows in a direction normal to the lines of forces of the electric ﬁeld and the mixture to be separated is added continuously at a small spot in the ﬂowing medium. Components of the mixture are deﬂected in diagonal trajectories according to their electrophoretic mobility and can be collected at the bottom of the chamber into as many as 96 fractions. Free-ﬂow electrophoresis (FFE) was born as a technique for purifying cells and sub-cellular organelles, which could be recovered highly puriﬁed as thin zones, due to their very low diﬀusion coeﬃcients. The Hannig apparatus went through successive designs and improvements, from an original liquid descending curtain to the present commercial version, dubbed Octopus, exploiting an upward liquid stream (Kuhn and Wagner, 1989). Instrumental to the success of the method, especially when run for long periods of times, is the constancy of the elution proﬁle at the collection port, so that the same protein species is always collected into the same test tube. Thus, electroendoosmotic ﬂow should be suppressed via a number of ways, including glasswall silanol deactivation and addition of polymers (e.g., 0.1% hydroxypropylmethyl cellulose), providing dynamic wall coating and proper liquid viscosity. FFE in a ProTeam apparatus was recently reported by Zischka et al. (2003) for puriﬁcation of S. cerevisiae mitochondria, previously puriﬁed by fractional centrifugation. These authors claimed identiﬁcation of many more proteins (n = 129) from FFE-puriﬁed 6 Righetti, Castagna, Herbert, and Candiano mitochondria as compared with mitochondrial protein extracts isolated by differential centrifugation (n = 80). In addition, a marked decrease of degraded proteins was found in the FFE-puriﬁed mitochondrial protein extracts, suggesting that the organelles were contaminated by lysosomes. FFE would not be ideal for protein pre-fractionation, though, due to their higher diffusion coefﬁcients, as compared with cells and organelles. However, Kobayashi et al. (2003) reported a microfabricated FFE device useful for continuous separation of proteins. Their separation chamber is barely 66 · 70 mm in size, with a gap between the two Pyrex glass plates of only 30 lm (see Fig. 1a and b). The liquid curtain and the sample are continuously injected from ﬁve and one holes, respectively, at the top. At the bottom of the chamber, a micromodule fraction collector, consisting of 19 stainless steel tubes, is connected perpendicular to the chamber and liquid stream. FFE, for protein separation, would work much better in the isoelectric focusing mode (FF-IEF), due to built-in forces impeding entropic peak dissipation. The ﬁrst report on the use of FF-IEF for pre-fractionation of total cell lysates from HeLa and Ht1080 cell lines, in view of a subsequent 2-DE map, is perhaps the one of Burggraf et al. (1995), who collected individual or pooled fractions for further 2-DE analysis. Hoﬀman et al. (2001) proposed FF-IEF as the ﬁrst dimension of a 2-DE map, the eluted fractions being directly analyzed by orthogonal SDS-PAGE. In turn, individual bands in the second SDS-PAGE dimension were eluted and analyzed by electrospray ionization, ion-trap MS. By this approach, they could identify a number of cytosolic proteins of a human colon carcinoma cell line. One advantage of FFIEF is immediately evident from their data: large proteins (e.g., vinculin, Mr 116.6 kDa) could be well recovered and easily identiﬁed; on the contrary, recovery of large Mr species has always been problematic in IPG gels. Weber et al. (2004) have also adopted FF-IEF for the eﬃcient separation and analysis of peroxisomal membrane proteins. Their success was documented by the detection of PMP22, the most hydrophobic and basic protein (pI > 10) of peroxisomal membranes. Perhaps pre-fractionation of proteins could also be attempted by IEF in the recently-revived vortex-stabilized, free-ﬂow electrophoretic device (Ivory, 2004; Tracy and Ivory, 2004). ROTATIONALLY STABILIZED FOCUSING APPARATUS: THE ROTOFOR Behind this remarkable invention by M. Bier, there is a long history going back to the doctoral thesis of Hjerte´n (1967): as he was trained in astrophysics, his apparatus was a ‘‘Copernican revolution’’ in electrokinetic methodologies. Hjerte´n was the ﬁrst one to propose electrophoretic separations in a free zone (i.e., in the absence of anticonvective, capillary media, such as polyacrylamide and agarose gel networks) but he had to ﬁght the noxious phenomenon of electrodecantation, induced by gravity. He thus devised rotation of the narrow-bore tubes used as electrophoretic chambers around a horizontal axis, mimicking celestial planet motions! This must have spurred the fantasy of Svensson-Rilbe, in those days a colleague at the Uppsala University, who ﬁnally described a large multi-compartment electrolyzer, capable of fractionating proteins in the gram range (Jonsson and Rilbe, 1980). The cell was assembled from 46 compartments, accommodating a total sample volume of 7.6 l, having a total length of 1 m, hardly user-friendly! Cooling How to Bring the ‘‘Unseen’’ Proteome to the Limelight Fig. 1. Scheme of the miniaturized FFE apparatus. Reservoir R1 is used for the electrophoresis buﬀer, whereas R2 (0.1 M aqueous NaOH ethanol, 50:50, v/v), R3 (0.01 M HCl) and R4 (80% aqueous ethanol) are used for washing cycles. S1 is the sample reservoir, P1 P3 are peristaltic pumps for pouring the solutions into the separation chamber, the electrode reservoirs and the sample port, respectively. D is a dumper, DRN a drain duct, PF is a fuse unit for excessive pressure. B: Scheme of the micromodule fraction separator (MFS). A cross-section of the separation chamber’s bottom with upper and lower parts of Pyrex glass is shown (from Kobayashi et al., 2003, by permission). 7 8 Righetti, Castagna, Herbert, and Candiano and stirring were aﬀected by slow rotation of the whole apparatus in a tank ﬁlled with cold water. Bier’s 50-year-long love aﬀair with preparative electrophoresis in free solution produced, as a last evolutionary step, a remarkable device, the Rotofor (Egen et al., 1988; Bier, 1998). A preparative-scale Rotofor is capable of being loaded with up to 1 g of protein, in a total volume of up to 55 ml. A mini-Rotofor, with a reduced volume of about 18 ml, is also available. The device is assembled from 20 sample chambers, separated by liquid-permeable nylon screens, except at the extremities, where cation- and anion-exchange membranes are placed against the anodic and cathodic compartments, respectively, so as to prevent diﬀusion within the sample chambers of noxious electrodic products. At the end of the preparative run, the 20 focused fractions are collected simultaneously by piercing a septum at the chambers’ bottom via 20 needles connected to a vacuum source. The narrow-pI range fractions can then be used to generate conventional 2-DE maps. This is the original approach described by Hochstrasser et al. (1991). In recent times, this methodology has taken another turn: the Rotofor is used directly as the ﬁrst dimension of a peculiar 2-DE methodology, in which each fraction is further analyzed by hydrophobic interaction chromatography, using non-porous reversed-phase HPLC (Zhu et al., 2003). Each peak collected from the HPLC column is then digested with trypsin, subjected to MALDI-TOF MS analysis and MSFit database searching. By this approach, Wall et al. (2000) have been able to resolve a total of ca. 700 bands from a human erythroleukemia cell line. It should be stated, though, that the pI accuracy of this methodology which is still based on conventional carrier ampholyte-isoelectric focusing, CA-IEF, is quite poor: it ranges from ±0.65 to ±1.73 pI units, a large error, indeed. On a similar line of thinking, Davidsson et al. (2001) have sub-fractionated human cerebrospinal ﬂuid and brain tissue, whereas Wang et al. (2002) have mapped the proteome of ovarian carcinoma cells. More recently, Xiao et al. (2004) have reported a novel application of the Rotofor, not just for fractionation of intact proteins in presence of carrier ampholytes, but for fractionation of peptide digests of an entire proteome (in this case, human serum) in an ampholyte-free environment. The peptides themselves would act as carrier ampholyte-buﬀers and create a pH gradient via an ‘‘autofocusing’’ process (with a caveat, though: the pH gradient will be quite poor, since only a few peptides have good buﬀering power and conductivity in the pH 5 8 range). THE GRADIFLOW The Gradiﬂow is a multi-functional electrokinetic membrane apparatus that can process and purify protein solutions based on differences of mobility, pI and size (Margolis et al., 1995; Horva`th et al., 1996). Its interfacing with 2-DE map analysis was demonstrated by Corthals et al. (1997), who adapted this instrument for prefractionation of native human serum and enrichment of protein fractions. In a more recent report (Locke et al., 2002), this device was also shown to be compatible, in the pre-fractionation of bakers yeast and Chinese snow pea seeds total cellular extracts, with the classical denaturing/solubilizing solutions of 2-DE maps, comprising urea/ thiourea and surfactants. Whereas, in the case of size fractionation, this can be achieved with polyacrylamide coated membranes at diﬀerent %T and %C for sieving of macromolecules in given Mr ranges, its use for separating approximate pI How to Bring the ‘‘Unseen’’ Proteome to the Limelight 9 fractions is more complex and has to adopt low-conductivity buﬀers, such as those devised by Bier et al. (1984), so as to allow reasonably high voltage gradients and relatively short separation times. It should be remembered, though, that the Gradiﬂow operates on the principle of binary fractionations, so that, in general, only two populations can be collected during each run, the so called ‘‘upstream’’ and ‘‘downstream’’ fractions. More recently, Bae et al. (2003) adopted the Gradiﬂow for pre-fractionation of alkaline proteins from Helicobacter pylori, although the terminology ‘‘extremely basic fraction’’ seems an exaggeration, considering that all the species identiﬁed by MALDI-TOF MS hardly reached pI values as high as pH 10. SAMPLE PRE-FRACTIONATION VIA MULTI-COMPARTMENT ELECTROLYZERS WITH ISOELECTRIC MEMBRANES We have already mentioned multi-compartment electrolyzers (MCE; Jonsson and Rilbe, 1980; Bier, 1998), as a class of instruments based on conventional IEF in presence of soluble, amphoteric buﬀers (carrier ampholytes, CA). However, the MCEs based on Immobiline membranes represent a quantum jump over the previous technique (Righetti et al., 1989, 1990, 1992). This method relies on isoelectric membranes, fabricated with the same acrylic monomers adopted in IPG fractionations (Wenger et al., 1987; Righetti, 1990). Advantages of such a procedure are immediately apparent: (i) such a device oﬀers a method that is fully compatible with the subsequent ﬁrst dimension separation in 2-DE maps, a focusing step based on Immobiline technology. Thus, protein mixtures harvested from the various chambers of this apparatus can be loaded onto IPG strips without any need for further treatment, in that they are isoelectric and isoionic; (ii) it permits harvesting a population of proteins having pI values precisely matching the pH gradient of any narrow (or wider) IPG strip; (iii) as a corollary of the above point, much reduced chances of protein precipitation will occur, as compared to loading onto a narrow IPG strip an unfractionated sample composed of a much wider pI spectrum (in the latter case, proteins non-isoelectric in the given pH range will massively precipitate towards the ends of the IPG strips, most often co-precipitating neighbouring species); (iv) due to the fact that only proteins co-focusing in the same IPG interval will be present, much higher sample loads can be operative, permitting detection of lowabundance proteins. The original apparatus, as miniaturized by Herbert and Righetti (2000), Righetti et al. (2001) and Herbert et al. (2004), is shown schematically in Fig. 2a. In this exploded view, two terminal electrodic chambers are used to block, in between, three sample chambers. Fig. 2b is a schematic diagram of the MCE for initial plasma fractionation. The four disks in the upper part are the isoelectric membranes inserted in between the various chambers. In this particular set-up, the couple pI 5.0 and pI 6.0 is used as a trap for capturing albumin. By properly exploiting this pre-fractionation device, Pedersen et al. (2003) have been able to capture and detect a large number of the ‘‘unseen’’ yeast membrane proteome. Figure 3 gives an example of the large number of membrane proteins detected, via this pre-fractionation protocol, in the pH 7 10.5 range, an interval that cartographers of the 16 century would have described as ‘‘terra incognita’’, devoid of any landmarks, and stamped inside the contour of the map the inscription: 10 Righetti, Castagna, Herbert, and Candiano Fig. 2. (a) Exploded view of the miniaturized multi-compartment electrolyzer operating with isoelectric membranes. An assembly with only 5 chambers is shown (3 sample chambers and the two termini electrodic reservoirs). (b) Schematic diagram of the MCE for initial plasma fractionation. The four upper disks represent the isoelectric membranes to be sandwiched in between each chamber (by courtesy of Proteome Systems). ‘‘hic sunt leones’’, just as they did with the African continent. These data fully misspell the notion expounded by Gygi et al. (2000) (see Introduction) that 2-DE maps cannot detect membrane and low abundance proteins (see also Herbert et al., 2003); the key for ferreting them out is to use appropriate pre-fractionation methods associated with concentration. A number of additional approaches have been described such as miniaturized devices (Zuo and Speicher, 2002) and the Rotofor accommodating isoelectric membranes (Shang et al., 2003). Zhu and Lubman (2004) have modiﬁed the IsoPrime device from Hoefer so as to lessen run volumes signiﬁcantly; additionally, the protein content captured in each chamber was further fractionated via non-porous reversed-phase HPLC. The notion that isoelectric membrane-based devices could How to Bring the ‘‘Unseen’’ Proteome to the Limelight 11 Fig. 3. Coomassie brilliant blue stained 2-D gels of the alkaline MCE fraction from a membrane preparation of log phase yeast. The alkaline fraction was separated in the ﬁrst dimension IPG using 2% ASB 14 detergent. The excess detergent has combined with SDS to form mixed micelles in the second dimension gel and caused the smearing observed in the low molecular mass part of the gel. The 2-D gel is the display of a 1.0 mg membrane protein preparation using an 11 cm pH 7 10.5 IPG for the ﬁrst dimension and GelChipTM 8 18% T second dimension gel. The gel is annotated with 237 proteins, representing 93 unique gene products (from Pedersen et al., 2003, by permission). not capture very high pI proteins has been recently misspelled by Lalwani et al. (2004a). These authors used high pI membranes fabricated with quaternary ammonium derivatives of cyclodextrins and poly(vinyl alcohol), cross-linked with glycerol-1,3-diglycidyl ether and demonstrated compatibility with catholytes as caustic as 1 M sodium hydroxide. By the same token, this same group (Lalwani et al., 2004b) has produced hydrolytically stable, low-pI isoelectric membranes from low-pI ampholytic components, poly(vinyl alcohol), and a bifunctional cross-linker, glycerol-1,3-diglycidyl ether. The low-pI ampholytic components used contain one amino group and at least two weakly acidic functional groups. These new, very lowpI isoelectric membranes have been successfully used as anodic membranes in isoelectric trapping separations with pH <1.5 anolytes and have been found to be a good replacement for the hydrolytically less stable polyacrylamide-based isoelectric membranes. Now the circle is closed and no one can any longer claim that IPGs cannot capture very low and very high pI proteins! Perhaps, though, one of the limiting steps in fractionating samples with these devices is the length of time needed to capture a given protein population in a given chamber, due to the sieving properties of the isoelectric membranes. A remedy to the 12 Righetti, Castagna, Herbert, and Candiano slow migration of proteins in MCEs due to the sieving effect of isoelectric membranes has been recently proposed by Cretich et al. (2003) who suggested using hydrogel beads, in lieu of membranes, as pI barriers sandwiched in between the various chambers. Although this approach greatly reduced the focusing time, it was plagued by the presence of electroendoosmotic ﬂow (EOF), as the beads did not provide a ﬂow-tight system. Aware of that, we designed new amphoteric beads, composed of ionic acrylamide derivative monomers co-polymerized within the pores of a central ceramic hard core, minimizing thus mass transfer resistance of proteins that are transiently adsorbed onto the beads (Fortis et al., 2005a). Additionally, these beads exhibit a much reduced EOF, thus permitting fast separations in MCE devices (Fortis et al., 2005b) with minimal liquid ﬂux from chamber to chamber. It is anticipated that isoelectric beads will ﬁnd a role in proteomic applications as a result of a rapid separation in miniaturized devices. An interesting variant to the use of the MCE apparatus could be a kind of direct 2D method, as depicted in Fig. 4, by which the surface charge fractionation, as obtained in the various MCE fractions, is coupled to size discrimination by running directly the content of each MCE chamber into an SDS gel. The bands eluted from the latter step would then be analyzed directly by MS (Cottingham, 2003). MINIATURIZED ISOELECTRIC SEPARATION DEVICES Although in the previous section we have mentioned ‘‘miniaturized’’ multichamber instruments, in reality these approaches still accommodate sizable sample volumes in each chamber, of the order of 0.5 2 ml. Smaller devices have been recently described. In one approach Tan et al. (2002) have built a device consisting in 96 mini-chambers (~75 ll each) arranged in eight rows. Neighbouring chambers in a given row are separated by short glass tubes (4 mm innerdiameter, ID, 3 mm long), within which isoelectric hydrogels of speciﬁc pH values are polymerized. During focusing, the device is sandwiched between blocks incorporating reservoirs for catholyte and anolyte. This device has been used not for the fractionation of proteins, but rather of their digests. With the described set up, however, some reservations are to be underlined; one of them is that peptides will collect mostly around acidic and basic pI values, leaving preciously little few (mostly those containing His residues) in the pH 5 8 range. In the 2-DE map displayed in Fig. 2a of Tan et al. (2002) the peptides deriving from the digest of four protein markers are visibly grouped into vertical pillars centered around the following pIs: 4.5, 6.0, 8.8 and 10.0. In another approach, Zilberstein et al. (2003) proposed parallel processing in isoelectric focusing chips. The main separation tool, here, is a dielectric membrane (chip) with conducting channels that are ﬁlled by isoelectric hydrogels of varying pH values. The membranes are held perpendicularly to the applied electric ﬁeld and proteins are trapped in the channels whose pH values are equal to the pI of the proteins. Further progress in these parallel, miniaturized devices has been reported by the same group (Zilberstein et al., 2004a, 2004b). In yet a third approach, a system called ‘‘Off-gel IEF’’ has been described by Ros et al. (2002). Just like the multi-compartment separation technique, the system has been devised for the separation of proteins according to their pI and for their direct recovery in solution without adding buﬀers or ampholytes. The principle is to How to Bring the ‘‘Unseen’’ Proteome to the Limelight 13 Fig. 4. Schematic diagram of a 2-D method interfacing MCE fractions with SDS-PAGE. Upper drawing: MCE instrument assembled with seven sample and two electrodic chambers (the numbers on top refer to the pIs of the various isoelectric membranes). Lower drawing: loading of the content of each chamber directly into SDS-PAGE gels. The bands resolved after the second step will then be analysed by MS (Courtesy of D. Speicher, Winstar Institute). place a sample in a liquid chamber which is positioned on top of an IPG gel. Theoretical calculations and modelling have shown that the protonation of an ampholyte occurs in the thin layer of solvation closed to the IPG gel/solution interface (Arnaud et al., 2002). Upon application of a voltage gradient, perpendicularly to the liquid chamber, the electric ﬁeld penetrates into the channel and extracts all charged species (those having pI values above and below the pH of the IPG gel), thus vacating them from the sample cup. After separation, only the globally neutral species (pI = pH of the IPG gel) remain in solution. In a further extension of this initial work, the system was improved and adapted to a multi-well device, composed 14 Righetti, Castagna, Herbert, and Candiano Fig. 5. Experimental set-up used to perform Oﬀ-Gel separations with multi-cup devices, composed of either 10 (a) or 22 (b and c) wells in diﬀerent pH intervals, as speciﬁed under the gels (from Michel et al., 2003, by permission). of a series of compartments of small volume (100 300 ll) and compatible with current instruments for separation (Michel et al., 2003) (see Fig. 5). PRE-FRACTIONATION ON SEPHADEX BEDS Contrary to all above-described methods Go¨rg et al. (2002), reported a technique of sample pre-fractionation with neutral beads of dextran (Sephadex) to isolate proteins by isoelectric focusing prior to 2-DE map analysis. This is in fact a rediscovery of the well known ‘‘Radola (1973, 1975) technique’’, described in the 70s. When the method was ﬁrst introduced it became quite popular and as a consequence commercial products were designed to ‘‘guillotine’’ out the entire Sephadex cake into 20 pieces after migration (for a more thorough description of the principle and the set-up, see Righetti, 1983). The focusing process is of course induced by the presence of carrier ampholytes, the Sephadex beads being exploited only as an anticonvective medium. Proteomic analysis that follows is not often compatible with the presence of carrier ampholytes, which are diﬃcult to remove. Mass spectrometry is, for instance, not compatible, since also carrier ampholytes would be detected and not easily distinguished from peptides of similar molecular masses. ACKNOWLEDGMENTS PGR is supported by FIRB 2001 (No. RBNE01KJHT), PRIN 2005 (MURST, Rome), Fondazione Cassa di Risparmio di Verona (Bando 2002) and by the How to Bring the ‘‘Unseen’’ Proteome to the Limelight 15 European Community (proposal No. 12793, Allergy Card, 2005). 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