A rapid microbore HPLC method for determination of primary structure of beta-lactoglobulin genetic variants

A rapid method for determination of the primary structures for beta-lactoglobulin (beta-LG) genetic variants is described. This included rapid microbore HPLC, amino acid analyses, and wherever necessary, direct peptide sequencing. Two novel variants of beta-LG have been identified, bovine beta-LG W and ovine beta-LG C. The proteins were oxidized, digested with trypsin and separated using RP-HPLC. All peptides were recovered in a single run. Peptides with amino acid exchanges were identified by retention time and subjected to amino acid and sequence analyses. Ovine beta-LG C differs from the ovine beta-LG A variant by a single amino acid exchange at position 148 where Arg is replaced by Gln. Bovine beta-LG W differs from bovine beta-LG B by having Leu at position 56 instead of Ile. The method described here is reliable and can be used for mapping of 20-1000 pmol of material.