Wheat cultivar discrimination by capillary electrophoresis of gliadins in isoelectric buffers

Abstract

A modified method is reported for screening of wheat cultivars: capillary zone electrophoresis of gliadins in isoelectric buffers. Previously published procedures recommended a 100 mM phosphate buffer, supplemented with 0.05% hydroxypropylmethylcellulose and 20% acetonitrile, in uncoated capillaries. Due to the very high conductivity of such a buffer (4.7 mmhos at 25 degrees C) high speed separations (10-12 min analysis time at 800 V/cm) could only be elicited in 20 microm internal diameter (ID) capillaries, at the expense of sensitivity. In the present report, we optimized the background electrolyte as follows: 40 mM aspartic acid (pH=pI=2.77) in the presence of 7 M urea and 0.5% short-chain hydroxyethylcellulose (Mn 27000 Da; apparent pH 3.9 in 7 M urea). As an alternative recipe, the same isoelectric buffer can be supplemented with a mixed organic solvent composed of 4 M urea and 20% acetonitrile (apparent pH 3.66). Due to the much lower conductivity (0.7 mmhos), separations can be carried out at 1000 V/cm in only 10 min, but in larger bore capillaries (50 microm ID), ensuring a five-times higher sensitivity. The gliadin patterns thus obtained are species-specific and allow easy identification of all cultivars tested of both durum and bread wheat. No adsorption of proteins to the silica wall seems to occur and high reproducibility in peak areas and transit times is obtained.

An N‐methylpolyvinylpyridinium cationic polymer for capillary coating in electrophoresis of proteins and peptides

“…First of all, it would appear that, for best performance, such cationic polymers, adsorbed onto the silica wall, should be coupled with a BGE made of isoelectric buffers, in particular Asp. The use of Asp had been in fact advocated long ago in the separation of wheat gliadins [36], of maize zeins [37] and of human globin chains [38]; while it appeared to quench interactions of proteinaceous analytes to the wall, it also allowed, due to isoelectric conditions, delivering high-voltage gradients to the silica tube, without generation of excessive joule heating. But there could be more to it: the fact of using amphoteric molecules, in particular a free amino acid, might help in quenching potential interactions of analytes with the silica wall.…”

Damaging effects of gliadin on three-dimensional cell culture model

“…Gliadin was purified from Triticum aestivum flour (Hereward cultivar) according to Capelli et al [12] . Bovine serum albumin (BSA) used as a control was purchased from Sigma (Milan, Italy).…”

Variety identification in maize linesvia capillary electrophoresis of zeins in isoelectric acidic buffers

“…Bossi and Righetti [22] also reported the use of imino diacetic acid (IDA) as an isoelectric buffer of low pI (2.23 at 100 mM concentration) and good solubility in water and in hydro-organic solvents. Isoelectric buffers have already been used for wheat cultivar discrimination via CZE of gliadins [23], and for separation of human globin chains [24]. A preliminary report on the separation of maize zeins has recently appeared [25].…”