Capillary electrophoresis of peptides and proteins in acidic, isoelectric buffers: recent developments

The use of isoelectric buffers in capillary zone electrophoresis is here re viewed. Such buffers allow delivery of very high voltage gradients (up to 1 000 V/cm in relatively large bore capillaries, e.g. 75-100 mu m I.D.), perm itting separations of the order of a few minutes and thus conserving (in fa ct favouring) very high resolution due to minimal, diffusion-driven, peak s preading. Isoelectric Asp (pl 2.77 at 50 mM concentration and 25 degrees C) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unre solved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta globin chains. Also imino diacetic acid (pl 2.33 at 50 mM concentration) allows generation of high resolution peptide m aps. Isoelectric Asp, in presence of 7 M urea and 0.5% hydroxyethyl cellulo se (Mn = 27 000 Da) is also the preferred medium for fast separation and an alysis of storage proteins in cereals, such as gliadins in soft and durum w heat and zeins in maize. A solution of 50 mM iminodiacetic acid (pI 2.23) c ontaining 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is effe ctively used as background electrolyte for fast separation of heme-free, de natured globin (alpha and beta) chains. In the presence of neutral to neutr al amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergen t micelle, via a mechanism similar to 'micellar electrokinetic chromatograp hy'. (C) 1999 Elsevier Science B.V. All rights reserved.