polyamine propane sultone r,-nh*--(ch2)„-nh2+-r2 + r.-nh+-(ch2)„-nht-r2
ch2 ch2 ch2
ch2 ch2 ch2
ch2 ch2 ch2
polyamine chloromethylenephosphonic acid hci + r,-nh+-(ch2)n-nh/-r2 + r-nh+-(ch2)„-nh*-r2
p032" p032" p032"
FIGURE 2.3. A: Chemical scheme for the synthesis of Ampholine. B: Chemical scheme for the synthesis of Servalyt. C: Hypothetical structure of a Pharmalyte constituent ampholyte containing six amines, one peptide bond, and three p-carboxylic groups. R corresponds to hydrophilic groups.
since it embraces the pI values of as many as 60% of all proteins expressed in living organisms. The findings: Ampholine contains 80 different Mr compounds, in the Mr interval 203 to 893 Da, for a total of 325 isoforms. Bio-Lyte consists of 66 different Mr species, in the Mr range 388 to 835 Da, for a total of 436 isoforms. Servalyt is made of 199 different Mr compounds, in the Mr interval 204 to 907 Da, for a total of 1302 isoforms. Pharmalyte pH 4-6.5 comprises 217 amphoteres, in the Mr range 150 to 1179 Da, for a total of 812 isoforms. Moreover, the vast majority of these species appear to focus as sharp zones, suggesting good buffering capacity and conductivity at pH = pI, in contrast with alkaline pH ranges (pH 8-10) where most species appear to focus poorly and thus exhibit rather shallow pH/mobility curves,
indicating poor buffering power and conductivity at pH = pi. This last observation is in agreement with findings from most scientists, who lamented severe difficulties in focusing basic proteins. At the light of these recent results, CA focusing in acidic pH ranges should be granted more credit for performances very close to those of iPGs. Additionally, although CA-IEF seems to have fewer followers in 2D map analysis, this technique seems to be quite valid in at least two important aspects of proteome analysis. In one instance, several groups have reported pre-fractionation of total cell lysates via preparative CA-IEF in a Rotofor instrument (or in a continuous free-flow electrophoresis device, such as the Octopus), enabling collection of up to 20 fractions to be further processed via 2D mapping and MS analysis (for reviews see references 23 and 24). In another experimental setup, CA-IEF is utilized, in a liquid vein, in a capillary format, followed by mobilization of the stack of focused proteins directly into and MS instrument . Here too better recoveries of proteins and a higher success in protein identification are claimed over gel-based approaches.
It had to happen, it was in the air. CA-IEF, the raging technique of the 1960s and 1970s, had begun to show the crippling diseases of age. Here is the list of the major problems lamented by users: a medium of very low and unknown ionic strength; uneven buffering capacity and conductivity; unknown chemical environment; not amenable to pH gradient engineering; cathodic drift (plateau phenomenon) . Some of these drawbacks were quite disturbing: for example, the fact that steady state, once reached, would quickly begin to dissipate, with drift of the entire stack of focused CAs (and proteins) toward both anode and cathode, would lead to irreproducible spot position in the final 2D map. The same would occur due to variability in CA production from batch to batch (the synthetic process being, by definition, chaotic!). The risk of complex formation between some proteins and some CA chemical structures was also lamented . When IPGs were first reported in 1982, it was apparent that all these problems had simply vanished .
IPGs are based on the principle that the pH gradient, which exists prior to the IEF run itself, is copolymerized, and thus insolubilized, within the fibers of a polyacrylamide matrix. This is achieved by using, as buffers, a set of six nonam-photeric weak acids and bases, having the following general chemical composition: CH2=CH-CO-NH-R, where R denotes either two different weak carboxyl groups, with pKs 3.6 and 4.6, or four tertiary amino groups, with pKs 6.2, 7.0, 8.5, and 9.3 (available under the trade name Immobiline from Pharmacia-GE). A more extensive set, comprising 10 chemicals (a pK 3.1 acidic buffer, a pK 10.3 basic buffer and two strong titrants, a pK 1 acid, and a pK > 12 quaternary base), is available as "pI select" from Fluka AG, Buchs, Switzerland. During gel polymerization, these buffering species are efficiently incorporated into the gel (84-86% conversion efficiency at 50°C for 1 h). Immobiline-based pH gradients can be cast in the same way as conventional gradient PAGs, using a density gradient to stabilize the Immobiline concentration gradient, with the aid of a standard, two-vessel gradient mixer. As shown in their formulas, these buffers are no longer amphoteric, as in conventional IEF, but are bifunctional. At one end of the molecule is located the buffering (or titrant) group, and at the other end is an acrylic double bond, which disappears during immobilization of the buffer on the gel matrix. The three carboxyl Immobilines have rather small temperature coefficients (dpK/dT) in the 10-25°C range, due to their small standard heats of ionization («1 kcal/mol), and thus exhibit negligible pK variations in this temperature interval. On the other hand, the five basic Immobilines exhibit rather large ApKs (as much as ApK = 0.44 for the pK 8.5 species) due to their larger heats of ionization (6-12 kcal/mol). Therefore, for reproducible runs and pH gradient calculations, all the experimental parameters have been fixed at 10°C.
Temperature is not the only variable that affects Immobiline pKs (and therefore the actual pH gradient generated). Additives in the gel that change the water structure (chaotropic agents, e.g., urea) or lower its dielectric constant, along with the ionic strength of the solution, alter their pK values. The largest changes, in fact, are due to the presence of urea: Acidic Immobilines increase their pK in 8 M urea by as much as 0.9 pH units, while the basic Immobilines increase their pK by only 0.45 pH unit. Detergents in the gel (2%) do not alter the Immobiline pK, suggesting that they are not incorporated into the surfactant micelle. For generating extended pH gradients, we use two additional chemicals that are strong titrants having pKs well outside the desired pH range. One is QAE (quaternary amino ethyl) acrylamide (pK > 12) and the other is AMPS (2-acrylamido-2-methyl propane sulfonic acid, pK 1.0). For the IPG run, the proteins are placed on a gel (or preferably adsorbed into the IPG strip during reswelling) with a preformed IPG. When the field is applied, only the sample molecules (and any ungrafted ions) migrate in the electric field. Upon termination of electrophoresis, the proteins are separated into stationary, isoelectric zones. Due to the possibility of designing stable pH gradients at will, separations have been reported in only 0.1 pH unit-wide gradients over the entire gel length leading to an extremely high resolving power (ApI = 0.001 pH unit, as opposed to a ApI = 0.01 pH for CA-IEF).
It should not be taken for granted that IPGs took over and gained popularity just after their invention: At the beginning, we only knew how to make narrow pH intervals (only 1-pH-unit wide) and the technique was scorned and taken as an ancillary device in comparison with CA-IEF. It took several years of modeling in order to understand how to make extended gradients, first 2-3 pH units wide and then, as more chemicals and strong titrants were produced, up to the most extended pH intervals, covering the grounds from 2.5 up to pH 11. We here report, in Figure 2.4, one of the most popular IPG gradients adopted in proteome analysis: a nonlinear pH 4-10 gradient. It is the only "democratic" gradient ever devised, since it gives equal space (thus equal rights) to the variegated population of proteins in any proteome: Given the relative abundance of different species (shown in the histogram), it is clear that an optimally resolving pH gradient should have a gentler slope in the acidic portion and a steeper profile in the alkaline region .
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