Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis

• Published in: Journal of chromatography. B 2006-03, Vol.833 (1), p.19-25
• Main Authors: Antonioli, Paolo, Mendieta, Martha E., Sebastiano, Roberto, Citterio, Attilio, Peltre, Gabriel, Busnel, Jean-Marc, Descroix, Stephanie, Candiano, Giovanni, Righetti, Pier Giorgio
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Buffers; Electrophoresis, Capillary - methods; Fractionation techniques; Fundamental and applied biological sciences. Psychology; General pharmacology; Hydrogen-Ion Concentration; Isoelectric buffer; Isoelectric Point; Medical sciences; Pharmacology. Drug treatments; Proteins - analysis; Ultra-narrow-pH ampholytes cuts; Uncoated-capillary CZE
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2005.10.017

Two different approaches are here reported for obtaining ultra-narrow p I cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise p I cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8–9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4–6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.