Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins.

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry.

Bioanalysis: Heri, hodie, cras.

This review, compiled for celebrating the decennial of the series of issues of Electrophoresis on bioanalysis, does not cover much heri (yesterday in Latin), since this was extensively described by my previous review Electrophoresis 2004, 25, 2111-2127, nor, for that matter cras (tomorrow in Latin) since making predictions on future scenarios is decidedly a nonscientific endeavor, but is concentrated onto hodie (today in Latin). Recent progress in the following fields is covered: SDS electrophoresis; IEF and the unraveling of the chemistry and composition of soluble carrier ampholytes; 2D maps; CE with microchip technology and some coatings of unique performance; chemistry and function of combinatorial peptide ligand libraries. This latter technique is becoming fundamental in the detection and identification of low-abundance proteins in proteome analysis.

Simulation of desalting that occurs during isoelectric trapping separations.

Simul 5, the simulation program based on the 1-D model of electrophoresis has been extended to simulate isoelectric trapping (IET) separations that take place in recirculating multicompartmental electrolyzers (MCEs). In the extended Simul 5, the simulated separation space between the anode and cathode can be divided into multiple segments to represent the anode compartment, separation compartment(s) and the cathode compartment. The compartments may have identical or different cross sections. A new algorithm simulates convective mixing that occurs in the recirculating MCEs where the distances between the buffering membranes are short and the velocities of tangential flows through the compartments, orthogonal to the electric field, are high. The intensity of simulated mixing can be independently controlled in each compartment. pH transients that were reported to occur during the desalting step in IET separations were simulated with the extended Simul 5 program: the main features of the experimental results were reproduced by the simulations. The simulations shed light on the possible causes of uneven anion and cation removal rates, pH transients and the transient invasion of the separation compartment by components of the electrode solutions that might occur during the desalting step.

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. IV: pH 6-8 intervals.

Four commercial brands of carrier ampholytes (Ampholine, Pharmalyte, Servalyt, Bio-Lyte), in the pH 6-8 range, have been analyzed by a 2-D technique based on preparative Rotofor fractionation followed by CE-MS of 10 out of 20 fractions harvested, in the second dimension. The findings: Ampholine (pH 6-8) contains 80 different M(r) compounds, in the M(r) interval 216-979 Da, for a total of 326 isoforms. Bio-Lyte (pH 6-8) consists of 62 different M(r) species, in the M(r) range 341-1048 Da, for a total of 237 isoforms. Servalyt (pH 6-8) is made of 126 different M(r) compounds, in the M(r) interval 240-785 Da, for a total of 703 isoforms. Pharmalyte (pH 5-8) comprises 123 amphoteres, in the M(r) range of 221-992 Da, for a total of 476 isoforms. Pharmalyte appears to be the best brand, with a good proportion of species focusing sharply at their pI position and relatively few 'poor' species, distributed along the entire pH gradient. General conclusions are drawn on the properties of all the pH intervals explored in this series of investigations and some guidelines for possible synthetic routes ameliorating the neutral and alkaline pH intervals are discussed.

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF. III: pH 2.5-4 intervals.

To study the molecular mass distribution and number of species in narrow-range (2-pH-unit wide, in the nominal pI 2-4 or 3-5 interval) carrier ampholytes from four commercial sources (Bio-Lyte, Servalyt, Ampholine and Pharmalyte), a 2-D technique was adopted, consisting of a preparative focusing step in a Rotofor instrument, followed by analysis of every other collected fraction (10 out of 20) by CE-MS. It was found that Ampholine pH 3.5-5 contains 105 different molecular mass (M(r)) compounds, in the M(r) interval 205-965 Da, for a total of 446 isoforms. Bio-Lyte pH 3-5 consists of 84 different M(r) species, in the M(r) range 216-965 Da, for a total of 383 isoforms. Servalyt pH 2-4 is made of 227 different M(r) compounds, in the M(r) interval 204-929 Da, for a total of 1201 isoforms. Pharmalyte pH 2.5-5 comprises 245 amphoteres, in the M(r) range 203-857 Da, for a total of 857 isoforms. Pharmalyte appears to be the best brand, with the vast majority of species focusing sharply at their pI position and almost no 'poor' species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the pI value. Due to some overlap with the adjacent acidic pH 4-6 interval, the species in common have been evaluated: the most extended overlaps are found in Ampholine (55% of the species appearing in the two neighbouring intervals) and in Servalyt (47% coincidence). The lowest overlaps are found in Pharmalyte (23%) and in Bio-Lyte (20%).

Mass distribution, polydispersity and focusing properties of carrier ampholytes for IEF II: pH 4-6 intervals.

For studying the M(r) distribution and number of species in narrow-range (2 pH-unit wide, in the nominal pI 4-6 interval) carrier ampholytes from four commercial sources (Bio-Lyte, Servalyt, Ampholine and Pharmalyte), a 2-D technique was adopted consisting of a focusing step in a liquid phase (Rotofor, yielding 20 fractions) followed by orthogonal CE in both, acidic and basic buffers. As a final step, every other fraction was analyzed by CE-MS. The findings: Ampholine contains 80 different M(r) compounds, in the M(r) interval 203 to 893 Da, for a total of 325 isoforms. Bio-Lyte consists of 66 different M(r) species, in the M(r) range 388 to 835 Da, for a total of 436 isoforms. Servalyt is made of 199 different M(r) compounds, in the M(r) interval 204 to 907 Da, for a total of 1302 isoforms. Pharmalyte pH 4-6.5, comprises 217 amphoteres, in the M(r) range 150 to 1179 Da, for a total of 812 isoforms. Pharmalyte appears to be the best brand, with the vast majority of species focusing sharply at their pI position and <5% "poor" species, distributed along the entire pH gradient, denoting an extremely shallow pH/mobility curve across the pI value.

Fructose-3-phosphate production and polyol pathway metabolism in diabetic rat hearts.

Previous studies have suggested that polyol-pathway and nonenzymatic glycation may be involved in the development of cardiac myopathy, a well-known manifestation of diabetes. Although the exact etiology of this complication is not fully understood, it is likely to be multifactorial. In this study, we investigated the metabolic consequences of diabetes and the effect of aldose reductase inhibitor (ARI) treatment on cardiac tissues of Sprague-Dawley rats. Perchloric acid (PCA) extracts of hearts from the animals were examined using 31P-nuclear magnetic resonance (NMR), gas chromatography/mass spectrometry (GC/MS), and high-performance liquid chromatography (HPLC). In 31P-NMR spectra of diabetic animals, a peak resonating at the chemical shift of 5.8 ppm with a coupling constant of 10 Hz was identified as fructose-3-phosphate (F3P). Undetectable in controls (< approximately 20 nmol/g), this metabolite was present at a concentration of 81.3 +/- 16.3 nmol/g wet weight (n = 4) in diabetic rat hearts. GC/MS analysis of these extracts from diabetics also identified a decomposition product of F3P, 3-deoxyglucosone (3DG), at a concentration of 9.4 +/- 3.5 nmol/g (n = 3), compared with 0.98 +/- 0.43 nmol/g (n = 3) in controls. No evidence was found for the expected detoxification products of 3-DG, 3-deoxyfructose and 2-keto 3-deoxygluconate. Concomitant with the elevation of F3P and 3DG, fructose and sorbitol levels were also elevated in diabetic animals. Surprisingly, ARI treatment was found to have no effect on the levels of these metabolites. These data suggest that either the heart may be unique in its production of fructose or it may not readily transport the ARI sorbinil. Production of the potent glycating agents F3P and 3DG in diabetics suggests that these compounds may be contributing factors in the glycation of cardiac proteins in the diabetic rat heart.

Direct detection of carrier ampholytes in immobilized pH gradients using picric acid precipitation.

A protocol is described for monitoring the heterogeneity of end products of organic syntheses yielding amphoteric molecules containing two or more amino groups. This protocol was found to be a valuable aid in synthesis of carrier ampholytes for specific isoelectric focusing applications. This method does not depend on the ampholytes themselves to dictate the conditions under which they are analyzed. Carrier ampholytes have been found previously to be insoluble in picric acid and the insolubility property was not dependent upon the pI of individual ampholyte species. This insolubility property was exploited in the protocol. Immobilized pH gradients were used to focus the carrier ampholytes. Ampholytes were then visualized in situ by picric acid precipitation. The data shows that the protocol is useful for analyzing the results of chemical manipulations for enhancing the resolution of carrier ampholytes. A direct relationship was shown between carrier ampholyte heterogeneity as demonstrated by this protocol and the resolution of complex protein mixtures in isoelectric focusing gels. Picric acid formed visible precipitates with a variety of organic compounds which contained more than one amino group.

Characterization of synthetic carrier ampholytes by repetitive scanning of the electric field during focusing.

This paper reports the utilization of a potential gradient array detector for monitoring the dynamics of the electric field during isoelectric focusing. Transient and steady state electric field profiles are presented for synthetic carrier ampholyte mixtures with a wide (approximately 3-10) pH range. Two available commercial products (Ampholine and Pharmalyte) and a laboratory synthesized mixture (PEHA ampholytes) are compared. The formation of conductivity gaps and their migration toward the cathode in extended experiments (cathodic drift) can be visualized with this system.

The buffer value of weak acids and bases: origin of the concept, and first mathematical derivation and application to physico-chemical systems. The work of M. Koppel and K. Spiro (1914).

A translation is offered of a paper by M. Koppel and K. Spiro, published in 1914, and entitled: 'On the action of moderators (buffers) in the shift of the acid-base equilibrium of biological fluids'. In this work, which appeared in Vol. 65 of the Biochemische Zeitschrift, the authors introduced the modern concept of buffer value and, for the first time, quantified the buffering power of weak acids and bases, ampholytes, dibasic acids, and mixtures of buffers. The circumstances that surround the eclipse of this trailblazing research have been critically examined, and biographical details have been provided.