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boundary is seen in bluish and the lower boundary somewhat reddish.

Conversely, in the focusing system, the two proteins are seen separated by 1-cm distance. Thus, it would appear that the present system has the potential of separating zones with molecular masses differing by as little as <1%, considering that a mass difference of 7.6% allows for about 1-cm free gel space and assuming a 1-mm free gel space between two adjacent zones to be an acceptable resolving power. This unique resolving ability was obtained by running our focusing system not in the presence of a charge gradient, as customarily run, but in the presence of a plateau of charges (a gel section with constant concentration of 12 mM pK 10.3 Immobiline), this leading to differential charge neutralization along the migration path.

12 mM

12 mM

ResolvingGel Tris-TricineSDS-PAGE

12 mM

12 mM

ResolvingGel Tris-TricineSDS-PAGE

Figure 5.6. Separation of a mixture of myoglobin and β-lactoglobulin conventional SDS-PAGE (right strip) vs. resolving gel (left strip, 4% T polyacrylamide gel with a 12 mM constant plateau of pK 10.3 Immobiline). The Mr markers are prestained. Red fraction (1) myoglobin; blue zone (2), β-lactoglobulin.

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it adopted buffers comprising a series of weak acids and weak bases, all monoprotic, but certainly non-amphoteric. The violation of Svensson-Rilbe’s law was only apparent, though: when these buffers were incorporated into the polyacrylamide matrix, and thus insolubilized, each infinitesimal gel layer perpendicular to the migration path was indeed amphoteric, isoelectric, and endowed with strong buffering power. Our present method represents the third way of performing focusing, and thus, perhaps, it closes the circle. In our system, we move from fully soluble buffers (conventional IEF) and fully insoluble species (IPGs) to a hybrid system, in which half of the charges are insolubilized (the basic Immobilines) into the polyacrylamide matrix, whereas the other half (the counterions) are soluble species. In the amphoteric, “isoelectric” complex, thus, a hybrid system, partly soluble, partly insoluble is formed.

5.4.1. ON THE SEPARATION MECHANISM

The novel method of SDS-PAGE focusing presented here offers some unique features worth discussing. First, it is of interest to speculate on the mechanism of separation. The pictogram of figure 5.1 gives an idealized vision of such a mechanism. Along the migration path, from cathode to anode, the polyacrylamide matrix exhibits an increasing concentration of positive charges, grafted onto the neutral matrix backbone, much like IPG matrices do, except that in the latter case the negatively charged, titrating counterion is grafted as well. As the negatively charged SDS-polypeptide chain complex migrates down the electrophoretic path, it encounters a linearly increasing density of positive charges. It is likely that, once a polypeptide chain of a given size meets along the path a density of positive charges matching its negative surface charges, there will be charge neutralization and the macromolecule will come to a stop. This explains the odd migration behaviour, by which, in a sieving matrix, the smaller polypeptides are the ones that are first arrested, whereas the larger peptides/proteins keep migrating toward the anode in regions of higher positive charge densities. Clearly, the smaller polypeptides have a lower total surface charge as compared to larger polypeptide chains, thus their migration is arrested, via this mechanism of charge neutralization, shortly after exiting from the application pocket. As demonstrated 168

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with the time course experiment performed with different charge gradients, the latter should be always chosen on the basis of the specific protein mixture, because larger proteins require higher charge densities in order to reach a final steady state, but the resolution of two neighbouring species is inversely proportional to the gradient steepness. The gradient may also be cast with a starting Immobiline concentration different from zero, so as to obtain shallow gradients capable of separating quite large proteins with small mass differences.

At the charge matching point, it is quite likely that the polypeptide chains, especially the largest ones, will distort locally the polyacrylamide fibers, attracting them to their surface, so that charge neutralization will occur efficiently. In fact, ion-to-ion interactions are among the strongest ones in non-covalent links among macromolecules [32]. In this regard, only monovalent ions were used as electrolytes, so as to minimally interfere with the ion-ion interactions and ensure proper focusing of the SDS-protein complex. Given the fact that we use rather dilute matrices (down to only 4% T) endowed with large pores, such local fiber distortion has to occur, since the average pore size will be of the order of a few nanometers. Additionally, gels are considered as viscoelastic media, fluctuating in the surrounding space [33], and this is especially true for dilute matrices, thus such local fiber distortion is a most likely event. The fact that our system works on a focusing principle is also implicit on the fact that the polyacrylamide matrices, being highly diluted, are minimally sieving; thus, they could not arrest the migration of small polypeptides via an approach to a pore limit, as customary in porosity gradient gels [34, 35].

There are other advantages of the present system worth mentioning. First there’s the fact that the relationship between Mr and migration distance is a linear function (see figure 5.3) as opposed to a semilog or even double-log function in conventional SDS-PAGE. The replacement of the logarithmic scale with the linear scale provides significant advantages, such as improved accuracy of mass determination and the ability for optimizing the separation for different mass regions, including very low and very high molecular mass ranges. Moreover, such a novel method could also permit estimation of the total surface charge of a given SDS-protein complex. For example, in the case of Figure 5.2, when measuring the distance migrated into the gel by, for example, β-lactoglobulin, and by

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assuming a 90% incorporation of the basic Immobiline into the polyacrylamide gel fibers, full protonation of almost all positive charges (given a pK of 10.3 and a running buffer pH of 8.3), and minimal pK variation of the free and bound Immobiline (as verified in practice by Bjellqvist et al., [36]), from its steady-state position one could calculate that the total negative surface charge molarity of the SDS-β-lactoglobulin complex is 9 mM, matching the molarity of Immobiline in the surrounding gel fibers. By the same token, for the smallest polypeptide insulin, one can calculate a negative surface charge molarity of its SDS complex of 3 mM.

To our knowledge, conventional SDS-PAGE could not give this type of information on the migrating protein-SDS complexes.

5.4.2. INNOVATIVE APPLICATIONS IN PEPTIDE ANAYSIS

Another interesting aspect of the SDS-PAGE focusing system is that it opens up a window for simple and efficient separation of tryptic digests and peptides in general, as shown in Figures 5.4 and 5.5. From this point of view, it is remarkable that a quite complex tryptic digest could be sharply separated in a strongly dilute polyacrylamide matrix. This has the advantage of allowing for easy extraction of the peptides for further analysis, for example, by mass spectrometry. This is really important indeed, because “peptidomics”, a branch of proteomics devoted to peptide fractionation and analysis, is now considered as central in biomarker discovery, since it is believed that a host of markers for diseases could be fragments of normal proteins abnormally processed during the onset of pathological conditions [37]. Moreover, with the discontinuous configuration presented here, one could easily select a molecular mass window of interest without any need for sample pre-fractionation, thus making the entire study faster and easier.

Another interesting aspect is the possibility of greatly modulating the migration distance of closely spaced proteins by a method that we have called “resolving gel”, as illustrated in figure 5.6. Here a constant charge plateau was used instead of a gradient of charges as typical of the present methodology. In a properly selected charge plateau, the difference of migration between two proteins with small differences in Mr values is magnified as the protein-SDS complexes are 170

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progressively titrated along their migration toward the anode. This method is not a steady-state, focusing method, as is customary of the present technique, but represents a case analogue to the “nonequilibrium IEF” of O’Farrell et al. [38], adopted for focusing in very alkaline regions where the lack of suitable carrier ampholytes impeded reaching a true steady state. The possibility of fine-tuning the present method to the separation of closely related species greatly enhances the flexibility of our technique, since it could, in principle, allow for separation of species differing by as little as 150 Da in their Mr values, a resolving power that is at least 1 order of magnitude greater than that exhibited by conventional SDS-PAGE.