Capillary electrophoresis of peptides and proteins in isoelectric buffers: An update

Abstract

Capillary electrophoresis in acidic, isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and little interaction of macromolecules with the untreated wall occurs. In addition, due to the low conductivity of quasi-stationary, isoelectric buffers, high-voltage gradients can be applied (up to 800 V/cm) permitting fast peptide analysis with a high resolving power due to minimal diffusional peak spreading. Four such buffers are here described: cysteic acid (Cys-A, pI 1.85), iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). A number of applications are reported, ranging from food analysis to the study of folding/unfolding transitions of proteins.

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

“…A Tris-Glycine buffer was used as the running buffer (10 mM Tris, 192 mM glycine, pH 8.5). This buffer is ideal for experiments involving capillary electrophoresis because it has a low absorbance at 214 nm, and has a low conductivity (which allows it to be subjected to high voltages without overheating the apparatus) (Hjerten et al 1995;Righetti et al 2000).…”

“…To determine whether any loss of activity was reversible, the solutions of BLA were cooled to 25°C for 1 h at the end of the 520-min, 90°C incubation, and the activity was measured. We chose to use Tris-glycine buffer because of its strong buffer capacity at alkaline pH (many enzyme-surfactant systems operate at alkaline pH) and also because Tris is an ideal buffer for electrophoretic experiments (Hjerten et al 1995;Righetti et al 2000).…”

High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteins

“…Isoelectric and phosphate buffer systems have been used to separate maize proteins. A review of various applications of CE to peptide/protein analysis in isoelectric buffers has been written by Righetti et al [72]. The capillary column most frequently used in the analysis of maize proteins has been an uncoated column with IDs ranging from 25 to 75 lm.…”

Capillary electrophoresis of proteins 1999-2001

“…Four acidic isoelectric buffers have been used in CE of proteins: cysteic acid (pI 1.85), iminodiacetic acid (pI 2.23), aspartic acid (pI 2.77) and glutamic acid (pI 3.22) [5,56].…”