Generation of tryptic maps of α- and β-globin chains by capillary electrophoresis in isoelectric buffers

Abstract

A novel method for generating peptide maps, following tryptic digests of proteins, is reported here: capillary zone electrophoresis in the presence of isoelectric buffers as the sole buffering species. A typical buffer composition comprises 50 m M aspartic acid (pH=p I=2.77), 0.5% hydroxyethyl cellulose (added as a dynamic coating agent for preventing peptide adsorption to weakly ionized silanols), 5% trifluoroethanol and 1% zwitterionic detergent (CHAPS). With this buffer composition, a high-voltage gradient can be applied (typically 600 V/cm in 75 μm I.D. and 900 V/cm in 50 μm I.D. capillaries), thus drastically reducing the analysis times. The method is applied to the generation of peptide maps of α- and β-globin chains from human adult hemoglobin. In the case of β-peptides, at an operative pH of 2.77, which represents a cross-over point in the titration curve of peptides T2 and T9, the two analytes merge into a single peak. However it is shown that it is possible to change the pH of the zwitterionic buffer by adjusting its concentration in solution. In 30 m M Asp (pH 3.0) or 20 m M Asp (pH 3.1) resolution of these two peptides is fully restored. Isoelectric, amphoteric buffers thus seem to represent a novel, powerful buffer system able to offer high resolution and high selectivity.