Two-dimensional peptide mapping by reversed-phase column chromatography, applied to the sequence determination of cytochrome c from the wild type and a mutant of the butterfly, pieris brassicae

Abstract

Two-dimensional peptide mapping has been very effective in the characterization of protein digests, particularly for the detection of small structural differences between homologous proteins. The classical thin-layer strategy, which exploits differences in charge and hydrophobicity, has been realized as a method based on reversed-phase high-performance liquid chromatography. An initial fractionation at pH 7.2 with 100 mM potassium phosphate, followed by chromatography with 0.1% trifluoroacetic acid, has been applied to chymotryptic digests of cytochromes c. The use of UV-transparent and (in the final stage) volatile solvents allows detection and rapid recovery of nanomole amounts of peptides suitable for sequence determination. As an example of the application of this method we report the comparison of two variants of cytochrome c from the butterfly, Pieris brassicae, one being the wild type and the other a spontaneous mutant isolated from a laboratory colony. The single residue difference was easily detected and identified.